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Effects of Khella (Ammi visnaga) Plant Extract on in Vitro Crystallization of Calcium Oxalate Monohydrate

Permanent Link: http://ufdc.ufl.edu/UFE0021623/00001

Material Information

Title: Effects of Khella (Ammi visnaga) Plant Extract on in Vitro Crystallization of Calcium Oxalate Monohydrate
Physical Description: 1 online resource (141 p.)
Language: english
Creator: Daosukho, Saijit
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Kidney stone patients are often given aqueous extracts of the Ammi visnaga (Khella) plant in many middle and near eastern countries. The mode of action of Khella as a kidney stone therapy is not well understood. We postulated that components of the extract may inhibit crystallization of calcium oxalate (CaOx), the major component of most kidney stones, and prevent crystal retention within the kidneys. This study was carried out to learn the composition of the Khella extract and investigate the effect the extract has on crystallization of CaOx in vitro. The composition of the plant extract was determined by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Concentration of the free ions in the extract was determined by oxalate assay kits and inductive couple plasma (ICP). Crystal induction time in supersaturated CaOx solutions was determined at 37 ?C using UV-VIS spectrometry. The induction time was estimated from the time vs. absorbance curve. Using an equation that relates induction times and supersaturation ratios, the surface energy, nucleation rate, free energy barrier, and critical nuclei radius were calculated. The interaction between free calcium and oxalate ions was determined by calcium titration. Aromic force microscopy (AFM) was used to study crystal-cell interaction between calcium oxalate monohydrate (COM) crystals and Marvin Darby Canine Kidney (MDCK) cells. The COM crystal was used in AFM measurements because the monohydrate structure is the most harmful to kidney epithelial cells. Khella was obtained from two sources, one in Turkey and one in Egypt. The HPLC and TLC results showed that only Turkish Khella extract contained khellin and visnagin which are believed to be the active components of the herb. The results from ICP and oxalate determination kits showed that both extracts contained calcium, magnesium, and oxalate. The plant extract reduced the induction time at every supersaturation ratio. From the induction time data, free energy barrier and critical nuclei radius were estimated. The calculation revealed a decrease of free energy barrier and critical nuclei radius as supersaturation ratio increased. From the calcium titration experiments, it was determined that the addition of Khella extract maintained the amount of free calcium ions in the solution. Scanning electron microscopy (SEM) images showed that the control supersaturated CaOx solutions produced CaOx monohydrate (COM) crystals. With the addition of Khella, the resulting crystals were of the CaOx dihydrate (COD) form. The slope of the light absorbance measurement curve indicated the inhibition of calcium oxalate nucleation from Khella extract. AFM measurements showed no negative interaction force between COM crystal and MDCK cells therefore, the addition of Khella extract reduced the chance of COM crystals adhere to kidney epithelial cells. The effect of individual components of the Khella extract, calcium, magnesium, khellin and visnagin, on calcium oxalate crystallization was also studied. All individual components identified, khellin, visnagin, calcium, and magnesium had no significant effect on kidney stone prevention in comparison to the extract. Therefore, we believe there are some unidentified components or a combination of components in Khella extract that are involved in the inhibition of kidney stone formation. Khella extract caused an increase in crystallization induction time, a change in the type of calcium oxalate crystal produced (COM to COD), inhibition effects for both nucleation and aggregation of calcium oxalate crystals, and a non-adhesive interaction to kidney epithelial cells. COM crystals are considered more injurious than COD crystals. The efficacy of Khella extract as a therapy for stone disease may be a result of its effect on the crystallization of CaOx.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Saijit Daosukho.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Svoronos, Spyros.
Local: Co-adviser: El-Shall, Hassan E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021623:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021623/00001

Material Information

Title: Effects of Khella (Ammi visnaga) Plant Extract on in Vitro Crystallization of Calcium Oxalate Monohydrate
Physical Description: 1 online resource (141 p.)
Language: english
Creator: Daosukho, Saijit
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Kidney stone patients are often given aqueous extracts of the Ammi visnaga (Khella) plant in many middle and near eastern countries. The mode of action of Khella as a kidney stone therapy is not well understood. We postulated that components of the extract may inhibit crystallization of calcium oxalate (CaOx), the major component of most kidney stones, and prevent crystal retention within the kidneys. This study was carried out to learn the composition of the Khella extract and investigate the effect the extract has on crystallization of CaOx in vitro. The composition of the plant extract was determined by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Concentration of the free ions in the extract was determined by oxalate assay kits and inductive couple plasma (ICP). Crystal induction time in supersaturated CaOx solutions was determined at 37 ?C using UV-VIS spectrometry. The induction time was estimated from the time vs. absorbance curve. Using an equation that relates induction times and supersaturation ratios, the surface energy, nucleation rate, free energy barrier, and critical nuclei radius were calculated. The interaction between free calcium and oxalate ions was determined by calcium titration. Aromic force microscopy (AFM) was used to study crystal-cell interaction between calcium oxalate monohydrate (COM) crystals and Marvin Darby Canine Kidney (MDCK) cells. The COM crystal was used in AFM measurements because the monohydrate structure is the most harmful to kidney epithelial cells. Khella was obtained from two sources, one in Turkey and one in Egypt. The HPLC and TLC results showed that only Turkish Khella extract contained khellin and visnagin which are believed to be the active components of the herb. The results from ICP and oxalate determination kits showed that both extracts contained calcium, magnesium, and oxalate. The plant extract reduced the induction time at every supersaturation ratio. From the induction time data, free energy barrier and critical nuclei radius were estimated. The calculation revealed a decrease of free energy barrier and critical nuclei radius as supersaturation ratio increased. From the calcium titration experiments, it was determined that the addition of Khella extract maintained the amount of free calcium ions in the solution. Scanning electron microscopy (SEM) images showed that the control supersaturated CaOx solutions produced CaOx monohydrate (COM) crystals. With the addition of Khella, the resulting crystals were of the CaOx dihydrate (COD) form. The slope of the light absorbance measurement curve indicated the inhibition of calcium oxalate nucleation from Khella extract. AFM measurements showed no negative interaction force between COM crystal and MDCK cells therefore, the addition of Khella extract reduced the chance of COM crystals adhere to kidney epithelial cells. The effect of individual components of the Khella extract, calcium, magnesium, khellin and visnagin, on calcium oxalate crystallization was also studied. All individual components identified, khellin, visnagin, calcium, and magnesium had no significant effect on kidney stone prevention in comparison to the extract. Therefore, we believe there are some unidentified components or a combination of components in Khella extract that are involved in the inhibition of kidney stone formation. Khella extract caused an increase in crystallization induction time, a change in the type of calcium oxalate crystal produced (COM to COD), inhibition effects for both nucleation and aggregation of calcium oxalate crystals, and a non-adhesive interaction to kidney epithelial cells. COM crystals are considered more injurious than COD crystals. The efficacy of Khella extract as a therapy for stone disease may be a result of its effect on the crystallization of CaOx.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Saijit Daosukho.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Svoronos, Spyros.
Local: Co-adviser: El-Shall, Hassan E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021623:00001


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1 EFFECT OF KHELLA ( Ammi visnaga ) PLANT EXTRACT ON IN VITRO CRYSTALLIZATION OF CALCIU M OXALATE MONOHYDRATE By SAIJIT DAOSUKHO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Saijit Daosukho

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3 To my Mom and Dad

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4 ACKNOWLEDGMENTS I would like to thank everyone who supported me during my years in graduate studies. I am very grateful to my advisor, Dr. Hassan El-Shall, who provided the greatest help, support, and understanding through all of difficulties during my whole graduated study. Without his encouragement and guidance, I would have a totally different life. I thank my other advisor, Dr. Spyros Svoronos who guided me through my tran sition in chemical engineering PhD program. Moreover specials thanks to my external committee member, Dr. Saeed Khan, for his help explaining biological matters regarding kidney stone subjects. I would also like to thank other members of my committee, Drs. Richard Dickin son and Yider Tseng in the Department of Chemical Engineering. I am grateful for the help on plant extraction and HPLC experiments from Dr. Butterweck and her gr oup. I would like to thank Dr. Yakov Rabinovich for his help with AFM measurement and interpretation, a nd a hearty thanks to Dr. Khans group, specifically, Karen Byer and Pa t Glenton, and the PERC staff. I am appreciated the help and discussion fr om my fellow graduate students in Dr. ElShalls group, Dr. Stephen Tedeschi, Rhye Hamey, and Kerri-Ann Hue I thank my good friends, Dr. Sukitti and Pawinee Punak, Theer apat and Pipawin Leesamphand, Dr. Kornvika Pimukmanaskit, Noppun Wongkittikraiwan and the Sartinoranont family for their help and mental support through out my years at the University of Florida. Hearty thanks to Shirley Kelly, Cynthia Sain, and Deborah Sandoval at the Ch emical Engineering Department, who have provided superb help getting me through all the pa per work. Special thanks to my families, who have always been the greatest supporter with love, care and encouraging words. Finally, I am grateful to the Particle Engi neering Research Cent er and the Industrial Partners of the ERC and the Thai Governme nt for financially s upporting this research

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5 TABLE OF CONTENTS Page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............14 CHAPTER 1 INTRODUCTION..................................................................................................................17 2 LITERATURE SURVEY.......................................................................................................19 Kidney Stone Disease........................................................................................................... ..19 Urinary Tract and Kidney Structure.......................................................................................19 Composition and Structure of Kidney Stones........................................................................23 Urine Composition and Its Role in Kidney Stone Formation.................................................25 Crystallization of Calcium Ox alate in Biological Systems....................................................26 Nucleation Theory...........................................................................................................26 Crystal Growth................................................................................................................30 Crystal Aggregation and Dispersion...............................................................................31 Crystal-crystal interaction........................................................................................31 Crystal-cell interaction.............................................................................................34 Treatment of Kidney Stone.....................................................................................................38 Extracorporeal Shockwave Lithotripsy...........................................................................38 Oral Supplements............................................................................................................39 Significance of Herbal Medici nal Treatment of Kidney Stone..............................................40 Herbs Related to Kidney Stone Treatment......................................................................40 Ammi visnaga (Khella) Extract.......................................................................................41 Characterization Techniques..................................................................................................43 High Performance Liquid Chromatography (HPLC)......................................................43 Inductively Coupled Plasma (ICP)..................................................................................44 UV-Vis Spectrometer......................................................................................................44 Atomic Force Microscopy (AFM)...................................................................................45 3 MATERIALS AND METHODS...........................................................................................47 Extract Preparation............................................................................................................ .....47 Extract Characterization....................................................................................................... ..47 Thin Liquid Chromatography (TLC)...............................................................................47 High Performance Liquid Chromatography (HPLC)......................................................48 Inductively Coupled Plasma (ICP)..................................................................................49

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6 Oxalate identification......................................................................................................50 Nucleation Study............................................................................................................... .....50 Induction Time Measurement..........................................................................................50 Membrane Isolation.........................................................................................................51 Solution Preparation........................................................................................................53 Calcium Titration Measurement......................................................................................54 Crystal Morphology and Crystallinity....................................................................................54 Crystal-Cell Interaction Study................................................................................................55 Preparation of COM Crystals for AFM Study................................................................55 Artificial Urine Solution Preparation..............................................................................56 Cell Culture................................................................................................................... ..57 Direct Force Measurement between COM Crystal and Kidney Renal Cells Using AFM............................................................................................................................ .58 4 RESULTS AND DISCUSSION.............................................................................................60 Khella Extract Active Com ponent Characterization..............................................................60 Inorganic Component Identification................................................................................60 Organic Component Identification..................................................................................60 Thin liquid chromatiography (TLC)........................................................................61 High performance liquid chromatography (HPLC).................................................61 Effect of Khella Extract on Ca lcium Oxalate Crystallization................................................64 Effect of Khella Extr act on Nucleation of Calcium Oxalate Crystals.............................64 Light absorption measurement.................................................................................64 Calcium titration measurement................................................................................67 Crystal morphology studies......................................................................................68 Surface energy and Gibbs free energy calculation...................................................73 Cell-retention of calci um oxalate crystal.................................................................81 Effect of Khella Extrac t Components on Calcium Ox alate Crystallization...........................83 Effect of Khellin and Visnagin on Calcium Oxalate Crystallization..............................84 Nucleation studies....................................................................................................84 Crystal morphology studies......................................................................................86 Effect of Calcium and Magnesium on Calcium Oxalate Crystallization........................87 Nucleation studies....................................................................................................87 Cell retention studies................................................................................................90 Effect of Other Urinary Species on Calcium Oxalate Crystallization....................................92 Effect of Citrate on Calciu m Oxalate Crystallization......................................................92 Nucleation studies....................................................................................................92 Cell-retention studies................................................................................................96 Effect of Cellular Membrane Debris on Calcium Oxalate Crystallization......................97 Effect of Urinary Proteins on Ca lcium Oxalate Crystallization....................................102 Nucleation studies..................................................................................................102 Cell retention studies..............................................................................................106

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7 5 CONCLUSION AND FUTURE WORK.............................................................................109 Conclusion..................................................................................................................... .......109 Future Work.................................................................................................................... ......113 APPENDIX A EFFECT OF KHELLA EXTRACT ON LI GHT ABSORBANCE MEASUREMENT......114 B EFFECT OF KHELLA ON THE CHANGE IN GIBBS FREE ENERGY OF FORMATION...................................................................................................................... .117 C AFM MEASUREMENTS OF COM CRYSTAL AND MDCK CELLS INTERACTION FORCE.......................................................................................................................... .......120 D EFFECT OF KHELLIN AND VIS NAGIN PURE COMP OUNDS ON LIGHT ABSORBANCE MEASUREMENT....................................................................................121 E ANOVA FOR % NUCLEATION INHIBITI ON RESPONSE SURFACE OF KHELLIN AND VISNAGIN PURE COMPOUND............................................................123 F EFFECT OF MAGNESIUM ON LIGHT ABSORBANCE MEASUREMENT.................125 G EFFFECT OF CITRATE ON LIGHT ABSORBANCE MEASUREMENT.......................127 H EFFECT OF CELLULAR MEMBRANE DEBRIS ON LIGHT ABSORBANCE MEASUREMENT................................................................................................................129 I EFFECT OF CELLULAR MEMBRANE DEBRIS ON CHANGE IN GIBBS FREE ENERGY OF FORMATION...............................................................................................132 J EFFECT OF ALBUMIN PROTEINS ON LIGHT ABSORBANCE MEASUREMENT...135 LIST OF REFERENCES.............................................................................................................137 BIOGRAPHICAL SKETCH.......................................................................................................141

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8 LIST OF TABLES Table page 3-1 Concentration of CaCl2 and K2C2O4 for each supersaturation ratio..................................53 3-2 Final concentration of each species test ed for light absorbance measurement..................53 3-3 Total concentration of artificial urin e displayed by the order of addition.........................57 4-1 ICP results for Egyptian and Turk ish extract freeze-dried flakes......................................60 4-2 The effect of Khella extract on induction time of calcium oxala te nucleation from UV-Vis light absorbance measuremen t at various supersaturations..................................65 4-3 New supersaturation ratio calculation of the system with Egyptian extract......................75 4-4 New supersaturation ratio calculation of the system with Turkish extract........................75 4-5 Effect of Khella extr act on surface energy of calcium oxalate crystals.............................75 4-6 Three level composites design experiment: th e effect of khellin and visnagin on light absorbance measurement...................................................................................................84 4-7 Effect of 0.1mM Mg2+ on induction time of calcium oxalate nucleation..........................87 4-8 Adhesion force between MDCK cells and COM crystal in Ca2+ and Mg2+ solutions.......91 4-9 The induction time estimation from light absorbance measurement with the presence of citrate..................................................................................................................... ........93 4-10 Effect of cellular membrane debris on th e induction time at various supersaturation.......98 4-11 The effect of cellular membrane de bris on surface energy of nuclei...............................100 4-12 Effect of albumin protein on inducti on time at various supersaturations........................103 4-14 Concentration of albumin and oxalate in AUIS solution used for AFM short range interaction force measurement.........................................................................................106 E-1 Analysis of variance table................................................................................................123 E-2 Statistic results.............................................................................................................. ...123 E-3 Diagnostics case statistics................................................................................................124

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9 LIST OF FIGURES Figure page 2-1 Structure of the human urinary tract..................................................................................20 2-2 Kidney frontal section schematic.......................................................................................21 2-3 Ultrastructural differences of the ma jor epithelial cells in nephron..................................22 2-4 Calcium oxalate crystal in three hydrate forms.................................................................24 2-5 The overall change in energy during nucleation................................................................27 2-6 Time-course measurements of OD620 (Optical Density at 620 nm) in a control experiment at standard conditions (calcium 5.0 mM, oxalate 0.5 mM)............................29 2-7 Electrical double layer.......................................................................................................32 2-8 The effect of ionic strength on the double layer................................................................33 2-9 Three possible conformations of adsorbed polymer on surfaces.......................................34 2-10 Calcium oxalate monohydrate crystal structur e model showing calcium-rich structure on (1 0 -1) face............................................................................................................... ....35 2-11 Changes in cell membrane phospholipids and nucleation of calcium oxalate...................36 2-12 Structure of khellin and visnagin.......................................................................................42 2-13 Atomic force microscopy...................................................................................................45 3-1 The TLC paper preparation................................................................................................48 3-2 Induction time approximation from UV-Vis light absorbance graph................................51 3-3 Layer separation of the brush border membranes isolation...............................................52 3-4 Instrument setup for mixing solution in the incubator to prepare COM crystals at 37 C.......................................................................................................................... ..56 3-5 An AFM tip attached with a single CO M crystal used for crystal-cell force measurement.................................................................................................................... ..59 4-1 HPLC graph of 0.5 mg/ml of pure standard khellin and visnagin.....................................61 4-2 Calibration curve for determination khellin and visnagin concentration from HPLC peak area...................................................................................................................... ......62

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10 4-3 HPLC graph of Egyptian Khella seed extract....................................................................62 4-4 HPLC graph of Turkish Ammi visnaga seed extract..........................................................63 4-5 Effect of Khella extract on light absorbance at initial supersaturation of 2.0....................65 4-6 Nucleation inhibitory effect of both Khella extracts..........................................................66 4-7 Effect of Khella extract on titration measurement of fr ee calcium ion in solution using calcium electrode at supersaturation 2.4..................................................................68 4-8 SEM picture of COM crystals formed at SS 2.8................................................................69 4-9 SEM image of the calcium oxalate crysta ls formed at SS 2.8 with 1 mg/ml of Egyptian Khella extract......................................................................................................70 4-10 SEM picture of calcium oxalate crystals formed at SS 2.8 with 1 mg/ml of Turkish Khella extract. ............................................................................................................... ....71 4-11 SEM picture of calcium oxalate crystals formed at SS 2.4 with 1 mg/ml of Turkish Khella extract................................................................................................................. ....72 4-12 Linear relationship between ln SS and ln Tind for the control solution..............................73 4-13 Induction time as a function of supersaturation plot used for surface energy calculation.................................................................................................................... ......74 4-14 Change in Gibbs free energy of formation with respect to supersaturation ratios.............79 4-15 Effect of Khella extract on GN at initial supersaturation of SS 2.0.................................80 4-16 Effect of Khella extracts on the change in critical free energy barrier at different supersaturation ratios.........................................................................................................81 4-17 Effect of Khella extract on COM crystals and MDCK in teraction force by AFM............82 4-18 Contour plot from statistical design on % nucleation inhibition of khellin and visnagin....................................................................................................................... .......85 4-19 SEM pictures of calcium oxalate crystals formed at SS 2.4 with 0.015 mg/ml Khellin and 0.0025 mg/ml visnagin................................................................................................86 4-20 Inhibitory effect of magnesium on calcium oxalate crystal nucleation.............................88 4-21 SEM picture of calcium oxalate crystals formed at SS 2.6 with the addition of 0.1 mM magnesium.................................................................................................................89 4-22 Calcium oxalate crystals in high calcium concentration....................................................89

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11 4-23 Effect of Ca2+ and Mg2+ on adhesion force between CO M crystal and MDCK cells.......91 4-24 Effect of citrate on light absorbance measurement at supersaturation 2.0........................93 4-25 Inhibitory effect of 0.1mM citrat e on calcium oxalate nucleation.....................................94 4-26 The effect of citrate on calcium oxa late monohydrate cr ystal structure............................94 4-27 EDS spectrometer of a single calcium oxa late crystal forming with citrate......................95 4-28 Effect of citrate on change in critical free energy barrier..................................................96 4-29 The interaction force measurement between COM crystal and MDCK cells in control (AUIS) and with 0.1mM of citrate in AUIS solution........................................................97 4-30 Effect of cellular membrane debris (MDCK and LLC-PK1) on light absorbance measurement at SS 2.0.......................................................................................................98 4-31 Effect of cellular membrane debris on nucleation rate inhibition......................................99 4-32 Effect of cellular membrane debris on ch ange in Gibbs free energy of formation at SS 2.2...................................................................................................................100 4-33 Change in critical free energy barrier as a function of supersaturation with the presence of cellular membrane debris..............................................................................101 4-34 Light absorbance measurement with the eff ect of albumin at supersaturation 2.0..........102 4-35 Effect of albumin on percent inhibi tion of calcium oxalate nucleation...........................103 4-36 Inhibitory effect of albumin on calcium oxalate crystals aggregation rate......................104 4-37 The effect of albumin on interaction fo rce between COM crysta l and MDCK cells......106 4-38 Short range interaction force/indenti on distance curve of MDCK cell and COM crystals....................................................................................................................... ......107 A-1 Light absorbance measurement with the presen ce of Khella extract at supersaturation 2.0.......................................................................................................................... ...114 A-2 Light absorbance measurement with the presen ce of Khella extract at supersaturation 2.2.........................................................................................................................114 A-3 Light absorbance measurement with the presen ce of Khella extract at supersaturation 2.4.......................................................................................................................... ...115 A-4 Light absorbance measurement with the presen ce of Khella extract at supersaturation 2.6.......................................................................................................................... ...115

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12 A-5 Light absorbance measurement with the presen ce of Khella extract at supersaturation 2.8.......................................................................................................................... ...116 B-1 Effect of Khella extract on GN at initial supersaturation of SS 2.0...............................117 B-2 Effect of Khella extract on GN at initial supersaturation of SS 2.2...............................117 B-3 Effect of Khella extract on GN at initial supersaturation of SS 2.4...............................118 B-4 Effect of Khella extract on GN at initial supersaturation of SS 2.6...............................118 B-5 Effect of Khella extract on GN at initial supersaturation of SS 2.8...............................119 C-1 Example of AFM measurements of COM-MDCK cells inter action in artificial urine solution with 1 mg/ml of Khella extract..........................................................................120 C-2 Example of AFM measurements of COM-MDCK cells inter action in artificial urine solution....................................................................................................................... ......120 D-1 Light absorbance measurement of standard # 5, 10, 11, 12, 13. (0.015 mg/ml Khellin and 0.0025 mg/ml visnagin)............................................................................................121 D-2 Effect of khellin on light absorbance measurement (standard # 1, 2, and 3)...................121 D-3 Effect of visnagin on light absorbance measurement (standard # 5, 1, and 4)................122 D-4 Comparison of light absorbance measur ement of all standard run number.....................122 F-1 Effect of magnesium on li ght absorbance measurement at supersaturation 2.0..............125 F-2 Effect of magnesium on li ght absorbance measurement at supersaturation 2.2..............125 F-3 Effect of magnesium on li ght absorbance measurement at supersaturation 2.6..............126 F-4 Effect of magnesium on li ght absorbance measurement at supersaturation 2.8..............126 G-1 Effect of citrate on light absorbance measurement at supersaturation 2.0......................127 G-2 Effect of citrate on light absorbance measurement at supersaturation 2.2......................127 G-3 Effect of citrate on light absorbance measurement at supersaturation 2.6......................128 G-4 Effect of citrate on light absorbance measurement at supersaturation 2.8......................128 H-1 Effect of cellular membrane debris on light absorbance measurement at supersaturation 2.0...........................................................................................................129 H-2 Effect of cellular membrane debris on light absorbance measurement at supersaturation 2.2...........................................................................................................129

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13 H-3 Effect of cellular membrane debris on light absorbance measurement at supersaturation 2.4...........................................................................................................130 H-4 Effect of cellular membrane debris on light absorbance measurement at supersaturation 2.8...........................................................................................................130 H-5 Effect of cellular membrane debris on light absorbance measurement at supersaturation 3.0...........................................................................................................131 H-6 Effect of cellular membrane debris on light absorbance measurement at supersaturation 3.2...........................................................................................................131 I-1 Effect of cellular membrane on the change in Gibbs free energy of formation at supersaturation 2.0...........................................................................................................132 I-2 Effect of cellular membrane debris on ch ange in Gibbs free energy of formation at supersaturation 2.2...........................................................................................................132 I-3 Effect of cellular membrane debris on ch ange in Gibbs free energy of formation at supersaturation 2.4...........................................................................................................133 I-4 Effect of cellular membrane debris on ch ange in Gibbs free energy of formation at supersaturation 2.6...........................................................................................................133 I-5 Effect of cellular membrane debris on ch ange in Gibbs free energy of formation at supersaturation 2.8...........................................................................................................134 J-1 Light absorbance measurement with the effect of albumin protein at supersaturation 2.0.........................................................................................................................135 J-2 Light absorbance measurement with the effect of albumin protein at supersaturation 2.2.......................................................................................................................... ...135 J-3 Light absorbance measurement with the effect of albumin protein at supersaturation 2.4.........................................................................................................................136 J-4 Light absorbance measurement with the effect of albumin protein at supersaturation 2.8.......................................................................................................................... ...136

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14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF KHELLA ( Ammi visnaga ) PLANT EXTRACT ON IN VITRO CRYSTALLIZATION OF CALCIU M OXALATE MONOHYDRATE By Saijit Daosukho December 2007 Chair: Spyros Svoronos Cochair: Hassan El-Shall Major: Chemical Engineering Kidney stone patients are often given aqueous extracts of the Ammi visnaga (Khella) plant in many middle and near eastern countries. Th e mode of action of Khella as a kidney stone therapy is not well understood. We postulated th at components of the extract may inhibit crystallization of calcium oxala te (CaOx), the major component of most kidney stones, and prevent crystal retention within the kidneys. Th is study was carried out to learn the composition of the Khella extract and investigate the eff ect the extract has on crystallization of CaOx in vitro The composition of the plant extract was determined by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Concentration of the free ions in the extract was determined by oxalate assay ki ts and inductive couple plasma (ICP). Crystal induction time in supersaturated CaOx solu tions was determined at 37 C using UV-VIS spectrometry. The induction time was estimated from the time vs. absorbance curve. Using an equation that relates induction times and supers aturation ratios, the surface energy, nucleation rate, free energy barrier, and cri tical nuclei radius were calculated. The interaction between free calcium and oxalate ions was determined by cal cium titration. Aromic force microscopy (AFM) was used to study crystal-cell interaction between calcium oxa late monohydrate (COM) crystals

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15 and Marvin Darby Canine Ki dney (MDCK) cells. The COM crystal was used in AFM measurements because the monohydrate structure is the most harmful to kidney epithelial cells. Khella was obtained from two sources, one in Turkey and one in Egypt. The HPLC and TLC results showed that only Turkish Khella ex tract contained khellin and visnagin which are believed to be the active components of the herb. The results from ICP and oxalate determination kits showed that both extracts contained cal cium, magnesium, and oxala te. The plant extract reduced the induction time at every supersatur ation ratio. From the induction time data, free energy barrier and critical nuclei radius were es timated. The calculation revealed a decrease of free energy barrier and critical nuc lei radius as supersaturation ra tio increased. From the calcium titration experiments, it was determined that th e addition of Khella extract maintained the amount of free calcium ions in the solution. Scanning electron microscopy (SEM) images showed that the control supersaturated Ca Ox solutions produced CaOx monohydrate (COM) crystals. With the addition of Kh ella, the resulting crystals were of the CaOx dihydrate (COD) form. The slope of the light absorbance measurem ent curve indicated the inhibition of calcium oxalate nucleation from Khella extract. AFM measurements showed no negative interaction force between COM crystal and MDCK cells theref ore, the addition of Khella extract reduced the chance of COM crystals a dhere to kidney epithelial cells. The effect of individual components of the Khella extract, calcium, magnesium, khellin and visnagin, on calcium oxalate crystallization was also studied. All individual components identified, khellin, visnagin, calcium, and magne sium had no significant effect on kidney stone prevention in comparison to the extract. Theref ore, we believe there are some unidentified components or a combination of components in Khe lla extract that are invo lved in the inhibition of kidney stone formation.

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16 Khella extract caused an increase in crystallization induction tim e, a change in the type of calcium oxalate crystal produced (COM to CO D), inhibition effects for both nucleation and aggregation of calcium oxalate cr ystals, and a non-adhesive intera ction to kidney epithelial cells. COM crystals are considered more injurious than COD crystals. The efficacy of Khella extract as a therapy for stone disease may be a result of its effect on the crystallization of CaOx.

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17 CHAPTER 1 INTRODUCTION Kidney stone disease, also known as Nephrol ithiasis and renal stones, is a common problem all over the world. It affects around 10% of the worlds population. The treatment of this disease costs over two b illions dollar per year [1-3]. Kidney stone formation begins when crystals of calcium oxalate, the main component of kidney stones, are retained or deposited insi de the kidneys, the crystals can grow or aggregate. The retained crystals block the urine flow leadi ng to increased pressure inside the kidney. This results in pain and discomfort. Less invasive methods beside surgery have been developed to treat kidney stone disease. One such method is extracorporeal shock wave lithotripsy (ESWL). ESWL uses shock waves to break kidney stones into small pieces that can easily pass through the urinary track[4]. Although, ESWL has been successful for treatment of kidney stone disease, many problems remain[1, 5]. Patie nts who undergo ESWL treatment may have increased incidences of hypertension and long-term renal damage. Stone fragments cannot be totally excreted from the kidney a nd may serve as sites for further nucleation and aggregation of crystals. ESWL is al so an expensive procedure for patients. Several chemical substances such as citrate, magnesium, and phosphate, taken orally, have been used to reduce the r ecurrence of kidney stones. However, these substances can cause some gastrointestinal si de effects[6-9]. Theref ore, an emphasis has been placed on the use of herbal medicinal tr eatments that are more cost efficient, are more effective, and have less side effects[10]. Herbal remedies, or folk medicines, are us ed in several parts of the world. In Egypt, Khella plant ( Ammi visnaga ) extract, used in the form of tea, has been found to stop the

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18 reoccurrence of the kidney st ones in the renal track[10, 11]. The plant extract has also been reported to help relax the ureter musc le, which reduces pain caused by the stones, and allows the stones to pass into the bladder more easily. However, the exact mechanism of Khella extracts active components on stone crystalli zation has not yet been clearly identified. The organic and inorganic component s of the Khella extract are suspected to play a role in inhibiting the nucleation and a ggregation of calcium oxalate crystals. These components are believed to affect either the calcium or oxalate ion concentration in the urine and therefore the calcium oxalate nucleat ion rate. Also, they might absorb onto the crystals surfaces and change the calcium oxalate crystal solubility. Kidney stones are composed of two phase s, inorganic crystals and an organic matrix. The diuretic mechanisms of Khella extracts active co mponents on kidney stone formation could effect both phases of the stone matrix. This project investigates the effect of Khella extract on the crystallization of ca lcium oxalate, a main inorganic component of kidney stones, in the presence and absence of other urinary species such as citrate and proteins.

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19 CHAPTER 2 LITERATURE SURVEY Kidney Stone Disease Kidney stone disease, nephrolithi asis or renal stone disease, is the most common urological disorder[1]. It is part of hu man biomineralization and refers to crystal deposition inside the kidneys. In the year 2000, approximately 2.7 mi llion patients had problems related to kidney stones and around 177,496 had admitted to hospitals in United States[3, 12]. The economic impact on healthcare related to kidney stone treatment was es timated to be $2.07 billion in 2000, in United States alone[12]. Around 80-90 percent of people who form kidney stones will have a reoccurrence of th e disease[1]. Kidney stones are formed from the ionic sa lt species found in urine. The most common biomineral forms of kidney stones are calcium oxal ate, calcium phosphate, a nd uric acid. In most cases, the crystals formed are very small and can pass through the kidney and urological system harmlessly. However, in some cases crystals aggr egate inside the kidney ca using the blockage of urine flow out of the kidney resulting in incr eased pressure, pain, and infection. Depending on the size and location, untreated kidney stones can cause permanen t damage to the kidneys[5]. Urinary Tract and Kidney Structure The human urinary tract consists of the kidneys ureters, bladder, a nd uretha (Figure 2-1). The kidneys work as a waste management cente r in the human body. They remove waste liquids, potentially harmful end products of metabolism, and extra water from the blood and convert them into urine. They also stabilize the essentia l substances in the blood such as water, sugars, amino acids, and electrolytes, as well as help produce hormones for building bones and generating red blood cells [3, 13]. The small tubes, called ureters, carry the urinary waste from

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20 the kidneys to the bladder where it is colle cted. The body excretes urine from the bladder through the uretha. Figure 2-1 Structure of the human urinary tract [5]. The kidney consists of two major regions, the medulla and cortex (Figure 2-2). The medulla is the inner section and is divided into small coned-shape sections called renal pyramids. Each renal pyramid contains a papilla. Urine fr om renal papillae passes into renal pelvis and from there into ureter. Each kidney in a human body contains roughly a million nephrons. The nephrons start in cortex act as small filtration units. They consis t of a glomerular capillary network surrounded by a tubular section called Bowmans capsule, a proxim al tubule, a loop of Henle, a distal tubule, and a collecting duct. Each segment of nephron or renal tubules has a different ultra structure (Figure 2-3).

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21 Co r te x M edu ll a Renal pyramids Renal pelvis Ureter Figure 2-2 Kidney frontal section schematic (taken form Grey Anatomy 1918)[14]. The waste products and excess water in the bl ood are forced pass the glomerulus junction by capillary force. Then reabsorbtion of the esse ntial salts, minerals and excess water begins throughout the tubular network. Each tubule is lined with of a singl e layer of different types of epithelial cells on the to p of a basal membrane. The tubular basal membrane provides the structural support for the epithelial cells and fo rms an uninterrupted framework throughout the length of the nephron. Various segments play si gnificant role in the formation of kidney stones[15]. The proximal convoluted tubule is where filtration starts and has a wider diameter than that of other sections involved in th e reabsorbtion process. The epithelium struct ure of the proximal segment has a brush boarder area with compact long and thin microvilli extensions. This structure provides high surface area to efficiently reabsorb important ionic salts back to the body

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22 (Figure 2-3a). The collecting duct is the last se ction of the nephron tubular system. End products and urine pass through the papillae, re nal pelvis and finally the ureter. Figure 2-3 Ultrastructural differences of the major epithelial cells in nephron. Schematic showing closed view of epith elial cell structure of a) proximal convoluted tubule and b) collecting duct[13]. The epithelial cells of the collecting duct segmen t are different from those in the proximal area. The cell brush boarder surface is composed of short microvilli that are less abundant and coarser than in the proximal se gment (Figure 2-3b). This structure provides a smooth surface to

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23 collect urinary waste but does not aid in reab sorbtion. Also, the length of microvilli structure changes the internal diameter of renal tubules. Therefore, the in ternal diameter of the proximal convoluted tubules is smaller b ecause of the longer microvilli, while the collecting duct has larger internal diameter because of shorter microvilli. These differences in the microvilli structure on the epithelial cell surfaces may infl uence calcium oxalate cr ystal retention in the kidney and may be related to the formation of kidney stones. Therefore, two cell lines that resemble these two sections of the urinary trac t have been used in most kidney stone related research[15-20]. The details of crystal-cell interaction will be discussed later on in the crystallization of calci um oxalate section. Composition and Structure of Kidney Stones Kidney stones are composed of organic and inorganic components [21, 22]. Approximately 98 wt% of a kidney stone is the crystalline inorganic content, 90% of which is made of calcium oxalate mixed with calcium phosphate. Calcium oxa late crystals in the monohydrate form are the most typically found. There exists three hydrat e forms of calcium oxalate; calcium oxalate monohydrate, calcium oxalate dihyd rate, and calcium oxalate tri hydrate. The morphologies of these crystal structures are shown in Figure 2-4. Calcium oxa late monohydrate (COM) is the most commonly found in stones because it is the most thermodynamically stable. Calcium oxalate dihydrate (COD), the metastable form, is also found and calcium oxalate trihydrate (COT) is rarely found in kidney stones. COT is be lieved to act as the pr ecursor of the COD and COM crystals[23]. The difference in structure of each hydrate form can affect the renal epithelial cell condition. COM has sharp edges that can physically sc rape the renal epithelial cells and result in cell injury while the ot her two hydrate structures have rounde r and smoother edges. In addition,

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24 the COM structure has a large oxal ate-rich face which has been f ound to be harmful to the renal epithelial cells[16, 20]. Theref ore, COM is the structure of focus in this study. Figure 2-4 Calcium oxalate crystal in three hydrate forms; a) calcium oxalate monohydrate (COM), b) calcium oxalate dihydrate (C OD), and c) calcium oxalate trihydrate (COT)[24, 25]. The organic matrix of the stone (around 2.5 %wt) consists primarily of small proteins with a molecular weight of 30,000 to 40,000 Da which may also include some larger molecure such as albumin, shredded epithelial cell membranes and th eir lipids, and other ur inary proteins[13, 26]. The presence of the organic matrix in the st ones suggests that hom ogenous nucleation of the stones is unlikely[15, 27]. Heterogeneous nucleat ion is more probable since urine is a very complex solution of water, ions, inorganic sa lts, small molecules, and macromolecules. The presence of these organic substances in urine can act as sites for hete rogeneous nucleation[27]. Some urinary macromolecules such as the Ta mm-Horsfall protein (THP), albumin and bikunin are also believed to play some role in stone crys tallization[28-34]. However, there is no certainty

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25 which protein(s) inhibits or promotes stone formation. The effects of macromolecules on stone crystallization depend on the ambient conditions in vivo Some proteins might have dual effects in crystallization by acting as promoter or i nhibitor depending on the urinary environment[30, 32, 33, 35]. Urine Composition and Its Role in Kidney Stone Formation Human urine is a naturally supersaturated solu tion of calcium oxalate. Therefore, it is normal for calcium oxalate crystals to nucleate in the urinary tract. As me ntioned in the previous section, beside calcium and oxalate, urine is also composed of other inorganic salt ions, small molecules such as citrate and phosphate, macromol ecules such as urinary proteins and lipids, and various metabolic wastes such as urea[36-38] The urine composition can be related to the formation of kidney stones, as these component s are present in both the organic matrix and inorganic crystalloids of the kidney stones[27]. Therefore, th e interaction between urinary species affects stone formation. Most stone fo rming patients were found to have an abnormal level of calcium and oxalate in the urine, which results in higher tendency in forming stone[26, 39, 40]. Higher from normal calcium concentration in urine is referred to as hypercalciuria. However, hypercalciuria may be difficult to iden tify since the excretion of calcium also depends on a persons diet and tolerable levels can na turally vary between patients. The excessive ingestion of calcium, e.g. drinking a lot of milk, or sodium may eff ect the excretion of calcium in the urine[26]. A high oxalate concen tration in urine is referred as hyperoxaluria. The majority of kidney stone patients are diagnosed with an abnormal oxalate metabolism[13]. Not only does hyperoxaluria increase nucleation of crystals, but, a high oxalate conc entration is associated with cell membrane injury [27]. When cells are exposed to a high con centration of oxalate either by the oxalate ion itself or as calcium oxalate crystals, the renal epith elia cells are injured and brush

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26 border membrane is shredded and sloughed into the urine. This cell debris can act as a site for heterogeneous nucleation. The injured cells also expose the basement membrane which could provide a binding site for COM crystals. Crystallization of Calcium Oxalate in Biological Systems Crystallization of calcium oxalate needs to be investigated to achieve a better understanding of the stone forma tion mechanisms. Several research ers have suggested that some urinary species such as citrate or proteins such as THP act as inhibitors of kidney stone formation. More details on those studies will be discussed later in this dissertation. The crystallization process can be categorized in to three main topics: nucleation, growth, and aggregation/dispersion. Nucleation Theory Nucleation is the first step of crystallization and supersaturation of the solution is a main driving force. Supersaturation occurs when the concentration of ionic sp ecies in solution exceed the saturation point. The supersaturat ion ratio (S) is calculated by[40]: *c c S (2-1) where c and c* is solute concentration and solute solubility at the given temperature. For nucleation to occur, the system has to overcome the energy barrier, Gibbs free energy of formation. The free energy of formation ( Gf) is given by the sum of two contributions, the change in surface energy and the change in volume energy according to the following[37, 41]: v fG r r G 3 23 4 4 (2-2) where is the surface free energy, in units of ener gy per unit area, r is the radius of a solid cluster, and Gv is the volume free energy. The plot of the free energy of formation as a function of radius is given in Figure 2-5:

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27 From the schematic in Figure 2-5, the free ener gy of formation rises to a maximum as the radius of a solid cluster, r, reaches the critical radius, r*, and then falls. This model suggests that before the solid clusters reach th e critical radius size, they form and redissolve continuously. After the critical size is reached, the clusters achieve th ermodynamic stability and become nuclei. This nucleation model e xplains only homogeneous nucleation theory where the formation of nuclei is based on su persaturation alone. Figure 2-5 The overall change in energy during nucleation. Based on the classic homogeneous nuc leation theory, induction time (tind) can be correlated with the supersaturation ratio (S) according to the following Equation: 2 3) (log ) log( S T B A tind (2-3)

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28 where A is an empirical constant (dimensionle ss) and B depends on a number of variables which are given below[40]. Plotting log of induction time versus 1/(log supersaturation) 2 gives a straight line with slope equal to B/T3, where T is the absolute temperature in Kelvin and 3 2 3) 3 2 ( ) ( R f N V BA m (2-4) Here is a geometric (shape) factor, 16 /3 for a spherical nucleus; f( ) is a correction factor, which is equal to 1 for homogeneous nucleation and equal to 0.01 for heterogeneous nucleation; Vm is the molecular volume, 66.418 cm3/mol for COM (74.6 cm3/mol For COD); T is the absolute temperature in Kelvin; R is the gas constant in J/mol K; is surface energy inJ/m2; and NA is Avogadros number in mol-1. The nucleation rate (Js), free energy barrier ( Gcr), and the radius of nucleus (r) can also be calculated using the following Equations: 2 3 2 3) (ln ) ( ) ( exp S RT f N V F JA m s (2-5) KT G F Jcr sexp (2-6) 23 / 4 r Gcr (2-7) where F is a frequency constant known as a preexponent factor, which has an empirical value of 1030 nuclei/cm3sec, and K is the Boltzman constant. Induction time has been used to measure a nd compare the crystallization of calcium oxalate crystals and the effect of other urinar y species on nucleation. El-Shall and his group used the relationship of induction time and supersaturati on ratio discussed above to identify the effect of citrate on COM nucleation[38]. They found that citrate does not sign ificantly change the surface energy of the COM crysta l nucleate but does retard the induction time by lowering the

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29 supersaturation through citrate complexes formed w ith the free calcium ions in the solution. The calculation of surface energy, nucle ation rate, free energy barrier a nd radius of nucleus has also been widely used in other system s such as calcium sulfate[42]. Hess also used induction time to explain the inhibitory effect of citrate and the TammHorsfall protein (THP) on COM crys tallization by measuring the ch ange of optical density over time[30]. Typical data can be s een in Figure 2-6. The inhibitory effect of citrate and THP was assessed by calculating the maximum slope of in crease of optical density with time as the maximum rate of nucleation, SN. The maximum rate of aggregation, SA, was identified by the highest negative slope of optical density. Figure 2-6 Time-course measurements of OD620 (Optical Density at 620 nm) in a control experiment at standard conditions (c alcium 5.0 mM, oxalate 0.5 mM). SN, is the maximum slope of the increase of OD620 with time, i.e. maximum rate of crystal nucleation and SA is the maximum slope of the decrease of OD620 with time, i.e. maximum rate of crystal aggregation [30].

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30 However, homogeneous nucleation rarely occurs in real biological systems like the urinary environment, and therefore nucleation is usua lly heterogeneous. Th e homogeneous nucleation theory discussed previously can be used to provide adequate information about the surface energy of the primary crystal nu cleation in the system. Heter ogeneous nucleation occurs on a foreign site present in the syst em, which allows the nucleation to occur at lower supersaturation levels. The foreign site in the urinary system can be either a pre-existing crystal, macromolecules such as urinary proteins, or cellular debris. Crystal Growth Every person will always nucleate calcium oxalate crystals inside kidneys due to high supersaturation of calcium and oxalate in urin e. For non-stone patients, the calcium oxalate crystals are small enough (less than ~ 2 m) to excrete out off nephrons without causing damage to the epithelial cells. Nucleation is necessary for st one formation, as it is the initiative step, but it is not the most important mechanism leading to kidney stone problems. In order for kidney stones to cause problems, crystal mass has to b ecome large enough to be retained inside the nephrons. The growth of crystals involves a m echanism of particle co arsening called Oswald ripening. This happens when ther e are a variety of particle sizes in the mixer and the larger particles grow at the expense of the smaller partic les in order to obtain a lower total energy of the system. Purely crystal growth is not believed to contribute much to the stone formation since the kidney filters urine at the rate of approximate ly around 180 L/day. The calcium oxalate crystals need to grow to the size bigger th an 2 m in less than 10 minutes to avoid getting excreted out of the body. This time interval is too short for normal crystal grow th, so we can conclude that growth is not the main mechanism in the stone fo rmation. Thus most research has been focused on the mechanism of calcium oxalate crystals aggr egation and how they ar e retained inside the kidney.

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31 Crystal Aggregation and Dispersion It has been stated that stone formation is init iated by a crystal that is retained inside the kidney and grows to a stone t oo large to pass thr ough the nephrons. Crystal aggregation is suspected to be a primary cause for kidney stone formation. Christ mas[37] concluded that of all urinary species, citrate concentration, oxalate concentration, and pr oteins were the most significant variables on crystal aggr egation. Fasano and Khan[15] also stated that the presence of membrane vesicles promotes crystal growth a nd aggregation which lead s to crystal retention within the kidney. The formation of a kidney st one also involves the dispersion of the calcium oxalate crystals. Urinary prot eins such as THP, bikunin, and osteopontin were among the macromolecules that have been s hown to inhibit stone formation by acting as a dispersants [43]. Also, several studies showed herbal kidney st one remedies involve in increasing crystal dispersion. Ammi visnaga extract is also suspected to effect the dispersion of calcium oxalate crystals in the urinary sy stem. To understand how the Ammi visnaga extract effects crystal aggregation and dispersion behavi or, the fundamentals of crystalcrystal interac tion and crystalcell interaction should be discussed. Crystal-crystal interaction The mechanism of crystals aggregation and di spersion can be explaine d using the particleparticle interaction theory. Particle-particle inte ractions influence the di spersive or aggregated state of a system. If the attractiv e forces between particles or crys tals dominate the overall forces in the system, aggregation occurs. On the other hand, dispersion or stabilization of the system occurs when the repulsive forces dominate. Several forces are involved in the particle-particle interaction and include electrost atic force, steric force, and van der Waals force[37, 43]. Electrostatic forces are developed when a soli d particle is immersed in a solution resulting in the formation of surface char ges. Surface charges are formed due to surface ion absorption,

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32 surface ion imbalance, defects, or ionic ex change and dissociation of surface acids. GouyChapman proposed a model of excess ionic char ges on the surface of a particle and surrounding ionic species in solution, called the electric double layer. The doubl e layer consists of the inner layer of charged ions opposite to the surface charge called Stern la yer, and the outer layer called diffuse layer (Figure 2-7). The dispersion of particles in solution depends on the sepa ration distance from the Stern layer due to the electrostatic re pulsion of the same charged laye rs. When particles are in a high ionic strength solution such as in urine solution, the Stern layer is comp ressed. The particles can then come close enough so that van der Waals force which is a short range attractive force overcomes the electrostatic repulsion force causi ng the particles to coagulate (Figure 2-8). Figure 2-7 Electrical double layer.

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33 Figure 2-8 The effect of ionic strength on the d ouble layer. a) At low ionic strength, the double layer separation is large. b) At high ionic strength, th e double layer is compressed. The steric force is also consid ered to be an important factor in the kidney stone aggregation mechanism. Steric force is the physical interaction force of each molecule in the atomic scale. Urine contains some macromolecules such as the urinary proteins and lipids of which some, as the Tamm-Horsfall protein (THP), are considered to have a dispersive effect on the calcium oxalate crystals while others, such as albumin, ar e considered to form polymer bridging leads to crystal aggregation. Steric interaction is depended on the surface coverage of the adsorb polymers or macromolecules. The possible confor mations of macromolecules that adsorb on the particle surface can be categori zed to three stages depending on surface coverage (Figure 2-9). When the surface coverage is low, polymers te nd to adsorb on surface with more than one segment causing the mushroom shaped conforma tion where the macromolecules cluster on the adsorbed surface with no overlapping to the othe r particle surface. When the surface coverage is high, more polymers concentrate on the surface forci ng the polymers chains to be close together and to extent the chain away from the surf ace thus causing the brush border conformations. These two conformations provide steric stabi lization to the system since the adsorbed macromolecules create longer rang e of surface separation that can overcome the van der Waals force and add electrostatic repulsion if the macr omolecules are of the same charge. However, if the surface coverage is not too low and too high, then polymer bridging between two surfaces

PAGE 34

34 can occur causing particle flo cculation by attaching th e other end of a polymer chain to the unoccupied surface site of the other particle[43]. Figure 2-9 Three possible conforma tions of adsorbed polymer on surfaces. a) Mushroom shaped conformation at low surface coverage with no overlap with another surface. b) Bush border conformation at high surface cove rage. c) Bridging between two surfaces when the coverage is not low or high.[44] The inorganic and or ganic species in Ammi visnaga extract could impact the crystal-crystal interaction mechanisms either by changing ionic st rength of the solution or changing the physical adsorbed surface. These species might complex w ith free ionic species in urine and increase the Stern layer separation distance. Or they could ad sorb on the calcium oxalate crystal surfaces and enhance the steric stabilizati on of calcium oxalate crystals. Crystal-cell interaction There is some suggestion that injured renal tu bular epithelia cells in kidneys are involved in the development of calcium oxalate crystal a ggregates due to the increased retention force between crystal and injured cell. Epithelia cells can be inju red by exposure to high oxalate concentrations or by direct scraping from sh arp calcium oxalate monohydrate (COM) crystals. When the cell is injured, it releases substances like renal prothrombin fr agment-1 (RPTF-1) or other anionic proteins/substances that induce the agglomeration of COM crystals leading to the formation of a stone[17]. Also, the injured cells tend to reveal the invert ed side of membrane,

PAGE 35

35 which is anionic, to the urinary environment. The anionic inverted membrane can act as the site for a COM crystal to adhere and grow[20, 31, 45, 46]. Verkoelen et al used the con-focal microsc ope to study the COM cr ystal attachment to Mardin-Darby Canine Kidney (M DCK) cell cultures[46]. The results show COM crystals binding during the development of a confluent monolayer of the MD CK cells. Also of interest, it was seen that when injuring the cell culture, th e COM crystals specifically bind to the area of injured cells during the wound healing process and none of th e COM crystals bind to uninjured cells. These results confirm the hypot hesis that the injured epithelia cells cause the retention of COM crystals inside the kidney and lead to the formation of kidney stones. Figure 2-10 Calcium oxalate monohydrate crys tal structure model showing calcium-rich structure on (1 0 -1) face [31].

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36 Figure 2-11 Changes in cell membrane phospholip ids and nucleation of calcium oxalate. A-a: Normal membrane with only neutral phos pholipids (yellow circ les) on the outer surface. B-b: Movement of acidic phospholip ids (red circles) from inside to the outside. C-c: Lateral movement of acidi c phospholipids into specific domains. D-d: Concentration of calcium ions and ca lcium oxalate nucleation on the acidic phospholipid domains [31]. Khan et al investigated the roles of lipids on the formation of kidne y stones[31]. In the study, they found that the crystallization of calcium oxalate monohydrate on a Langmuir monolayer depends on the influe nce of lipid headgroup charges. Langmuir monolayer is a one molecule thick layer of an in soluble organic material spread onto an aqueous subphase. Most COM crystals were observed at the interfaces with anionic headgroup (glycerol > serine

PAGE 37

37 choline). The binding mechanism of COM crysta l on the negatively charged interface can be explained by the attraction of the anionic regi ons to the free calcium ions in the solution by electrostatic force which resulted in regions of highly concentrat ed calcium. This concentrated calcium region induced the nucleation of COM cr ystal on the anionic lipid site and led to a crystal orientation with a calcium rich face on th e lipid surface. The COM crystal structure and nucleation mechanism on Langmuir monolayer is shown below. Figure 2-11 shows the schematic result when the cells are inju red and the involvement of calcium oxalate nucleation to the change in surface phospholipids asymmetry. When the cell is injured, the apical (outer) surf ace looses its asymmetry, which l eads to the exposure of anionic (or acidic) phospholipids from the basolateral (inner) membrane to the apical membrane. These acidic phospholipids then move to form a doma in which attracts the free calcium ion to concentrate above. The high calcium concentration due to supersat uration leads to nucleation of calcium oxalate crystals on the surface. Also, th e anionic phospholipid domain leads to a crystal orientation with a calcium-rich face along the phospholipids surface. Rabinovich et al used atomic force micros copy (AFM) to measure the interaction force between silicon nitride tips-cells and COM crystal-cells[18, 19]. Two renal epithelial cell lines were used, the Madin-Darby Canine kidney epithelial cell (MDCK) and proximal tubular epithelial cells derived from pig kidneys (LLC-PK1). MDCK cells were used to represent the collecting duct kidney epithelial cell and LLC-P K1 cells were used to represent the kidney proximal tubular epithelial cell, which are the two sections on which most calcium oxalate crystals are found. There was no attractive force between the AFM tip and cells. However, when attaching a COM crystal to the AFM tip and measuring the interact ion force between COM crystal and cells, they found an increased attr active force between the COM and MDCK cell

PAGE 38

38 while no sign of adhesion force between CO M crystal and LLC-PK1 cells. Also when investigating the interaction between COM crys tal and the basal membrane (BM) of both cell lines, similar results were seen. Only the BM of MDCK cells show adhesion forces with COM crystals. When studying the effect of oxalate tr eated cells on the COM cr ystal interactions, only MDCK cells showed an increase of adhesion fo rce with COM while the LLC-PK1 again showed no significant adhesion fo rce. These studies provide a possible explanation on the preferential deposition of COM crystal in the collecting du cts segment (represented by MDCK cells) and lack of COM crystal appearance in the proxi mal tubules parts in the animal models. Treatment of Kidney Stone Kidney stone removal surgery is currently considered a safe and effective procedure for treating kidney stone patients. However, more than 50% of kidney stone patients face the problem of kidney stone recurren ce and have to repeat the surg ery over and over again. Going through several surgical treatments to remove ki dney stones could be pain ful, frustrating, and costly. Several methods are developed in order to avoid kidney stone surgery for the patient. Extracorporeal shockwave lithot ripsy, oral supplements and herbal remedy are effective alternatives. Extracorporeal Shockwave Lithotripsy Extracorporeal shockwave lithot ripsy (ESWL) is a method using sound waves to break the kidney stone to small pieces. When the stone brea ks into small pieces, it ca n be excreted out of kidney safely without surgery. However, ES WL does not prevent the recurrence of kidney stones; therefore, patients who reform kidney stones have to go through ESWL repeatedly. The method of ESWL also involves a huge water-sonicated pool which is less convenient than taking oral supplements. Long term treatment of ES WL may cause hypertension and could lead to chronic kidney failure. The fragments of stone that remain in the ki dney could be sites for

PAGE 39

39 heterogeneous nucleation of new stones. Also, rep eated treatments of ESWL can be costly and time consuming. Oral Supplements Several oral supplements have been given to the kidney stone pa tients as an aid for preventing the recurrence of kidney stones. The well-known supplements for kidney stone treatment mostly contain citric acid and magnesium salt. Citrate is known to be one of the inhibitors of kidney stone formation. Citrate in human plasma exists as an alkaline citrate (C6H5O7 3-) in a range from 0.05-0.3 mmoles/liter[47]. The abnormal level of citrate in human urine, known as hypocitraturia, is one of major contributors to kidney stone disease besides hyperc alciuria and hyperoxaluria. H ypocitraturia is a symptom that urinary citrate is decreased from the normal le vel by enhanced reabsorb ance by renal tubules and impaired uptake[48]. Due to the anionic charge of alkaline citrate, it competitively binds free calcium ions in urine so that there are fewer avai lable free calcium ions to interact with oxalate ion and form calcium oxalate crystals. Th e reactions below show such mechanism. Ca2+ (aq) + C2O4 2(aq) + n H2O CaC2O4.nH2O(aq) Ca2+ (aq) + C6H5O7 3Ca3(C6H5O7)2 (aq) CaC2O4.nH2O(aq) CaC2O4.nH2O(s) The solubility of calcium citrate is higher than the solubility of cal cium oxalate in any hydration state. Therefore, the calcium citrat e complex stays in aque ous solution while the calcium oxalate aqueous complex pr ecipitates to solid crystals; thus citrate inhibits nucleation. Pak et al. showed that the treatment of hypocitraturia slow ed down or eliminated kidney stone formation [7, 49]. In vitro studies have shown citr ate to inhibit precipitat ion of calcium oxalate crystal [30, 38]. Also, citrate has been shown to prevent aggregation and growth of calcium oxalate [9].

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40 Potassium citrate has been given to kidney stone patients as an oral supplement to treat hypocitraturia by physicians because of the inhibitory effect of citrate stated above[50]. Sodium citrate is also used, however not as frequently since high sodi um intake may increase urinary calcium excretion. Magnesium salt is another main oral suppl ement used for kidney stone disease. Magnesium ion has the same possitive charges as calcium ion. Th erefore, magnesium ions competitively bind free oxalate ions the same way as citrate does to calcium ions. Several studies have proved the effectiveness of magnesium in preventing or reducing the recurrence of kidney stones in both rat and human subjects. Hamm arsten et al. studied the effect of MgCl2 on the solubility of calcium oxalate in water. They found out that addition of MgCl2 in the solution increases the solubility of cal cium oxalate[51]. Gershoff et al. showed that when adding magnesium in a kidney stone-induced rat subject, th e deposition of calcium oxalate crystal to the kidney was reduced[52]. Also they tested with 30 human stone former subjects and found out that MgO oral supplement helped reducing or eliminating the recu rrence of kidney stones. Leiske et al. studied the effect of magnesium on the interaction betw een calcium oxalate monohydrate crystals and kidney epithelial cells They measured the number of 14C COM crystals that stuck to cells after being treated with Mg2+ and Ca2+ ions and rinsed with buffer solution. They found that there were maximal adhesions of COM crystals to the MCDK cells at the critical concentration of Ca2+ and Mg2+, above the normal physiological concentration in urine[53]. Significance of Herbal Medicin al Treatment of Kidney Stone Herbs Related to Kidney Stone Treatment Herbal remedies of kidney stone have been known for many centuries and used all over the world. Also, herbal remedies or folk medicine are still widely practiced in the Mediterranean, Middle East, and Asia. Several plan t extracts have been identified as a diuretic and remedy for

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41 kidney stones. Atmani et al. investigated Herniaria hirsuta on calcium oxalate crystallization in vitro and in vivo [28, 54]. H. hirsuta has been widely used to trea t kidney stone symptoms in the Mediterranean area. Also in Japan, Alismatis rhizome, or Takusha, is used in traditional Japanese herbal therapeutics for the trea tment of stone disease [55]. Bergenia ligulata is used in India and Phyllanthus niruri in Brazil[56, 57]. Several investigators have studied the effect of the plants mentioned above on stone formation. Similar results have been seen in these studies. The extract induces the nucleation of the cal cium oxalate crystals; however, the crystals morphology is most often present in the di hydrate form instead of monohydrate form. Also the size of nucleated crystals becomes smaller as the plant extract concentration increases. Moreover, some plants extracts show dispersive activity on the calcium oxalate crystal leading to less aggregation. All studies show high potential of th e herbal extracts for the reduc tion of the stone reoccurrence, even though the plant extr acts may lead to the nucleation of more crystals. The generation of smaller, less stable calcium oxalate dihydrate crystals by the plant ex tract, leads to nephrons easily passing the crystals out the urinary tract. Ammi visnaga (Khella) Extract In addition to all of the pl ant extracts mentioned above, Ammi visnaga (Khella) is reported as an effective traditional remedy for kidney st one in Egypt as menti oned back in the Ebers papyrus (c 1500 BC)[10]. Ammi visnaga is a tall plant with thick stem and small leaves grown in the Middle East around the Medite rranean and in South Africa [58] The flower is white with seed inside. The extract is normally prepared by the infusion method or the decoction method to make herbal tea from dried seed. The herbal tea is given to the patients orally for a diuretic of kidney stone and to relieve pain related to the renal stone disease. The key constituents of Ammi visnaga are khellin, visnagin, khellol glycoside, volatile oil, fl avonoids, and sterols [10, 11]. Ammi visnaga extracts are also used as antispasmodic, antiasthmatic and muscle relaxant agents.

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42 The two key constituents of Ammi visnaga khellin and visnagin, were believed to be the active constituents of the plant [11]. Both khell in and visnagin are th e derivative form of coumarins, which has benzopyrone as a core skelet al structure. The structures of khellin and visnagin are shown in Figure 2-12. Figure 2-12 Structure of khellin and visnagin Khan Z.A. et al. concluded that a possibl e explanation for the diuretic activity of Ammi visnaga extract was because it prevents precipitati on of the calcium oxalate crystal, even though the concentration of oxalate is sufficien t. In the study, they also found that Ammi visnaga treated rats had less calculi depo sition in their kidney comp ared to the glycolic acid control group [59]. Rauwald et al. suggested th at the active compounds in Ammi visnaga fruits, khellin, visnagin, and visnadin could be involved with the Ca2+ channel blocking [60]. Ammi visnaga seeds also have an antispasmodic effect on the muscles wh ich reduces the pain from the trapped kidney stone and helps ease the stone down the bladder. From these studies of crystal-cell interaction, it can be seen that crystal retention inside the kidn ey is connected with th e cell injury mechanism. Ammi visnaga extract has been reported to ease the stone down to the bladder which means that the active constituents in the extract could also reduce the crystal-cell adhesion force or impact the releasing of substances during the cell injury itself.

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43 Characterization Techniques Characterization techniques used in this research include high performance liquid chromatography (HPLC), inductively coupled pl asma (ICP), light absorbance measurement by UV-Vis spectrometer, and atomic force micros copy (AFM) and these are discussed below. High Performance Liquid Chromatography (HPLC) High performance liquid chromatography is us ed to identify the presence of organic components such as khellin and visnagin in Khella extract. HPLC is developed from column chromatography, where high pressure is used to force liquid solution (mobile phase) through a column packed with stationary phase. Small volum e of the sample is injected through the column with a stream of mob ile phase. The separation of component s in the sample depends on physical and chemical interaction between the component, mobile phase, and the st ationary phase in the column. For each component, the time that it comes out at the end of the column is called retention time. Retention time is a unique characteristic for each specific component so it is used for identification methods. High pre ssure that is used in HPLC in creases the velocity of liquid flow through the column; therefore, it reduces re tention time and improves the resolution when compared with regular column chromatography and thin layer chromatography. The mobile phase composition can be varied during an anal ysis for higher resolution using a method called gradient elution. The gradient elution program controls the amount of each chemical in the mobile phase as a function of tim e. This allows the HPLC to se parate component s as a function of mobile phase composition which helps refine the resolution. The m obile phase composition depends on the nature, physical and chemical inte raction to stationary ph ase, of each component that needs to be separated. Mobile phases used for determination of khellin and visnagin in Khella extract by several research groups were mixture of water, acet onitride, tetrahydrofuran (THF), ethanol, and

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44 methanol in different ranges of concentrati on depending on the type of stationary phase of column. The composition of the mobile phase in this study is developed based on what has been done previously and the composition that maximi zed the retention time peak resolution is selected as described later in the material and methods section. Inductively Coupled Plasma (ICP) Inductively coupled plasma is used to dete rmine free ions of single elements such as calcium, potassium, and magnesium in Khella ex tract. The ICP spectrometer uses of an argon plasma torch at high temperature in the order of 10,000 K. When th e droplet of sample passes the plasma torch, the plasma evaporates any solid components that dissolved in the solution and breaks them down to atoms. The charge from the atoms from the sample is measured. The concentration of each single element ion in the sample is calibrated with the element standard solutions. UV-Vis Spectrometer UV-Vis spectrometer is equipment used to measure the absorption of ultraviolet and visible light after it passes thr ough a sample or after it reflects on a sample surface. To measure the nucleation, absorption of selected a single wave length is used and is measured as a function of time. The Beer-Lambert law provides the re lationship between analyte concentration and absorbance: bc A (2-8) where A is an absorbance, is the molar absorbtivity, b is the parth length of the sample, and c is the concentration of compound in solution. Therefore, the absorbance increases as a mixtur e nucleates crystals which reflect or absorb light. The absorbance is measured relative to a control solution in a cuvette. The solution with particles will have less light transmittance or higher light absorbance.

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45 Atomic Force Microscopy (AFM) Atomic force microscopy (AFM) is used to m easure the interaction force between a sample and an AFM tip on an atomic level. An AFM tip, the sharp end of a cantile ver, is normally made of silicon or silicon nitride. However, sometime the tip can also be modified by attaching a particle or polymer in order to measure the interaction force between a particle and a sample. The interaction force is measured by the movement of the cantilever that is caused by repulsive or attractive force between the tip and the samp le. The sample is placed on a piezoelectric scanner and then slowly rises to the AFM tip. Th e up or down movement of the cantilever due to repulsive or attractive force is detected by the m ovement of a laser path which points directly to the AFM tip. When the tip reflexes, it causes a laser beam to move and this movement is detected by a photo-detector. The schema tic of AFM is shown in Figure 2-13. Figure 2-13 Atomic force microscopy

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46 In this study, AFM is used to measure the interaction between a single COM crystal and kidney epithelial cell surface. A single COM crys tal is attached to an AFM tip and the measurement is done in a liquid ch amber. The urinary species are added to the buffer solution to study the effect of those species on crystal-cell interaction.

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47 CHAPTER 3 MATERIALS AND METHODS Extract Preparation Seeds were obtained from two sources, one in Egypt and one in Turkey. The Egyptian Khella seeds were obtained from herbalist in Egypt. Turkish seeds were obtained from Caelo (Caesar & Lorentz GmbH, Hilden, Germany) and certified to be Ammi visnaga Extracts were prepared from seeds of Khella by hot water infusion. 20 g of dried ground seeds were added to 200 ml of 100 C nanopure dei onized water. The mixtur es were left at 100 C for 5 minutes. The decoctions were left to co ol at room temperature. Following cooling, the mixtures were filtered through a 0.45 m membrane (Fisherb rand, Pittsburgh, PA) and the supernatants were kept. Both supernatants were freezed dried overnight. The dried flakes were collected and kept at 20 C for further use. The freeze-dried flakes of extract were collect ed and weighted to be approximately 3.5 g per 200 ml of extract decoction prepared. Then 0. 18 mg of freeze dried flakes of Khella extract were dissolved in 1 ml of deionized nanopure wa ter so that the final concentration for each experiment was equivalent to 1 to 100 dilution of the original ex tract decoction (1 mg of dried seed per 1 ml of deionized water). Extract Characterization Thin Liquid Chromatography (TLC) Organic components of the extr acts were identified using th in layer chromatography (TLC) and high performance liquid chromatography (HPL C). TLC was used as a quick screening on whether the extracts contained khelli n or visnagin or not. 0.5 mg of dried flakes were added to 5 ml of 60% ethanol. 5 mg of standard khellin and visnagin were added to 1 ml of 60 % ethanol. All solutions were filtered through 0.2 m f ilter paper (Fisherbrand, Pittsburgh, PA). The

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48 solutions were then transferred to TLC paper us ing capillary tubes. The TLC paper was marked at 1.5 cm line, so that the travel distance fo r the liquid chromatogram was 10 cm (Figure 3-1). After transferring all the solution at the 1.5 cm line using capillary tubes, the TLC paper was merged into ethyl acetate solution that ha d not reached the 1.5 cm drop marked. Then the TLC paper was left until the ethyl acetate m obile phase reached the 10 cm line marked. To identify khellin and visnagin in the extracts, the TLC paper was exposed to UVlight. Spots of khellin and visnagin in the extract should match the sta ndard khellin and visnagin. Figure 3-1 The TLC paper preparation. High Performance Liquid Chromatography (HPLC) HPLC was done to confirm the TLC results and quantify the amount of khellin and visnagin in the extract. The analysis was pe rformed using a Shimadzu liquid chromatograph (LC-10 AT) with a ternary solvent delivery sy stem, combined with a Diode Array detector (SPD-M 10 A), and a Rheodyne loading valve fitt ed with a 100 l sample loop. The HPLC

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49 column used for khellin and visnagin separation in Ammi visnaga extract was a LiChroCart RPselect (250 mm x 4 mm i.d., 5 m particle diameter) reversed-phase column, with a pre column (4.5 mm i.d. x 2.5 cm) containing the same packing. Separation of khellin and visnagin was done by using the gradient elution pr ogram and used the following solvents for the mobile phase: A was deionized water, B was Tetr ahydrofuran, THF, (Fisher Science, HPLC grade), and C was methanol (Fisher Science, HPLC grade). All solutions used for the mobile phase were filtered through a 0.45 m filter (Fisherbrand, Pittsbur gh, PA) and then degassed using an ultrasonicator. The gradient applied was as follows: 5 to 13% B from 0-20 minutes with constant 5% C and balanced with A, 13 to 22% B and 5 to 7% C from 20-52 minutes balanced with A, and then 22-5% B and 7-5% C from 5260 minutes balanced with A. 10 l of each sample, extract solutions, khellin standard and visnagin standard were injected and the detection of peaks was recorded at 330 nm (optimized wavelength). HPLC analyses were carried out at a constant temperature of 30 C. The mobile phase flow ra te was 1 ml/min. To qua ntify the amount of khellin and visnagin in the extract solution, se veral standardize mixtures of pure khellin and visnagin (Sigma Aldridge) in th e range of 0.05-1 mg/ml were used in the HPLC experiments to obtain a calibration curve. The amount of khellin a nd visnagin in the seed extracts was quantified using the areas of the peaks. Inductively Coupled Plasma (ICP) Calcium and magnesium ions in the extracts were identified us ing inductively coupled plasma (ICP, Perkin Elmer Optima 3200RL Optical Emission Spectroscopy, Norwalk, CT). The instrument was calibrated usi ng 0 ppm (nanopure water), 1 ppm, 10 ppm, and 100 ppm calcium (calcium reference solution cer tified 1000 ppm 1%, Fisher Scientific, Fair Lawn, NJ) and magnesium (magnesium referen ce solution certified 1000 ppm 1%, Fisher Scientific, Fair Lawn, NJ) solution and evaluated using non-linear regression. For each sample, 5 readings were

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50 taken at all wavelengths for calcium and magnesium defined in the program. The pump flow rate was 1 ml/min and delay time was 60 seconds. Oxalate identification Since oxalate is not single element components, ICP could not be us ed to analyze both components. To determine oxalate concentration of extracts, a modified microassay from an oxalate kit was used (591-D; Trinity Biotec hnology, St. Louis, MO). Briefl y, 100 l of extract was mixed with 100 l of sample buffer (supplied with kit). The 1:1 mix was then added to activated carbon in a 1.5 ml tube (supplied with kit). The tube was then mixed on a rotator for 5 minutes. The tubes were then centrifuged at 13000 rpm in a mi crocentrifuge. 10 l of clear supernatant was placed into a designated well of a 96 well plate. 10 l of 0.25 M, 0.50 M, and 1 mM oxalate standards (supplied with kit) were place in design ated wells. Reagent A was then added and then Reagent B (both supplied with kit) to all wells. The plate was gently shaken for 10 minutes at room temperature. The plate was read us ing a Bio-Rad Microplat e reader at 595 nm. Nucleation Study Induction Time Measurement The nucleation mechanism of calcium oxalate cr ystals can be determined by using optical density measurements. A UV-Vis spectrometer (Per kin-Elmer, Lamda 800) was used to measure the light absorbance as a function of time. The i nduction time, time that detectable crystal can be identified, was estimated from the point of ma ximum slope of light ab sorbance (Figure 3-2). A wavelength of 560 nm (optimized wavelength) was used to measure the change in light absorbance of each mixture in 4ml cuvettes. Th e light absorbance was measured every 5 seconds for 30 minutes. The UV-Vis spectrometer sample c uvette holder was attached to a heated water pump via rubber tubing to maintain th e sample temperature at 37 C.

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51 Figure 3-2 Induction time a pproximation from UV-Vis lig ht absorbance graph. The experiment was designed to study the eff ect of some of the ur inary species and of Khella extract and its component s on the induction time at differe nt supersaturation ratios of calcium and oxalate solutions. Th e supersaturation ratio was ca lculated based on Equation 2-1 which was given in Chapter 2. The range of supers aturation ratios was select ed to be 2.0 to 2.8. The induction time of other supersaturation ratios that were out of this range were obtained by extrapolating the data using linear relationship between natural l og of induction time and natural log of supersaturation ratio. Membrane Isolation The LLC-PK1 and MDCK cell membrane debris we re prepared and isol ated with the help of Karen Byers from Dr. Khans Pathology lab. The membrane isolation method used was based on the rate zonal sucrose gradient s according to a standa rd published protocol [61, 62]. The cells were homogenized with isola tion buffer (300 mM D-Mannitol, 5 mM EGTA, 12 mM Tris HCL, pH 7.4) using a handheld motori zed homogenizer (Fisher brand ti ssue tearor) for 10-20 seconds. 1.5 mL of 120 mM MgCl was added to the homogenized tissue mixture and shaken for 20 minutes on ice.

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52 Figure 3-3 Layer separation of the brush border membranes isolation. Then the mixture was centrifuged at 4500 rpm for 15 minutes. The supernatant and pallet were separated and saved. The pallet was homo genized and centrifuged again with the same method stated above. Then the supernatant was collected and combined with the supernatant from the previous spin. The supernatant was centrifuged again at 16000 rpm for 30 minutes. The pallet was collected after this spin. Then the pallet was homogenized again with 30 ml of strength isolation buffer. Then 3 mL of 120 mM MgCl was adde d to the mixture. The mixture was shaken for 20 minutes on ice then centrif uged at 4500 rpm for 15 minutes. The supernatant was collected after this spin. Then supernatant was centrifuged at 90000 rpm for 60 minutes at 4 C. After centrifugation, the supe rnatant was separated into layers containing different parts of cells (Figure 3-3). The layer of brush border membrane wa s gently pulled out using a micropipette. The content of memb rane in the solution was determined using a Lowry protein essay.

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53 Solution Preparation Solutions of calcium chloride (CaCl2, A.C.S. Grade, 74%-78% assay, Fisher Scientific Inc.) and potassium oxalate (K2C2O4, A.C.S. Grade, 100.8% assay, Fisher Scientific Inc.) were prepared at four times the concentration fo r each supersaturation ratio. For the control experiment 1 ml of calcium chloride, 1 ml of potassium oxalate, and 2 ml of deionized water were mixed together so the final concentration of the mixture matched the supersaturation ratio shown in Table 3-1 below. Table 3-1 Concentration of CaCl2 and K2C2O4 for each supersaturation ratio [CaCl2] [K2C2O4] Supersaturation Ratio mM mM 2.0 0.43 0.43 2.2 0.473 0.473 2.4 0.516 0.516 2.6 0.559 0.559 2.8 0.602 0.602 Table 3-2 Final concentration of each specie s tested for light absorbance measurement. To study the effect of each urinary species, so lutions of each species were prepared in deionized water at concentration of four times the desired final con centration. Each run, 1 ml of calcium chloride, potassium oxalate, deionized wate r, and urinary species of interest were mixed in a 4 ml cuvette and tested for the change of light absorbance. The final concentration of each species is shown in Table 3-2. Species Concentration Unit Egyptian Khella extract 0.1 mg (dry seed)/ml (water) Turkish Khella extract 0.1 mg (dry seed)/ml (water) Khellin 0.03-0.12 mg/ml Visnagin 0.005-0.03 mg/ml Citrate/ [Mg2+] 0.1 mM MDCK cell debris 10 g/ml LLCPK cell debris 10 g/ml Bovine Serum Albumin 15 ppm

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54 The relationship between induction time and supe rsaturation ratios was used to calculate surface energy of nuclei and the free energy ba rrier based on the classical homogeneous nucleation theory mentioned in chapter 2. Calcium Titration Measurement To study the interaction of calcium and oxalate in solution, the amount of free calcium ions in solution was checked using the calcium electr ode attached to a TIM 856 Titration Manager Radiometer. The amount of calcium ion in solutio n was measured as the potential reading in voltage which was converted to concentration using a calibration curve. Higher voltage means there is higher amount of calcium in solution. The titration measurement was recorded as a function of time. The depletion of calcium in solution means that there is less free calcium available in solution due to pr ecipitation or calcium binding of the component of interest. Crystal Morphology and Crystallinity To check the effect of Khella extract on calcium oxalate mo rphology, the crystals formed after 30 minutes of light absorbance measuremen t were collected by filtering the mixture through 0.2 m filter paper (Fisherbrand, Pittsburgh, PA). The crystals were transferred to carbon tape and attached to -inch scanning electron microsc ope aluminum stages. The samples were coated with gold using a plasma sputtering system (IB2 Ion Coater, Eiko Engineer ing) for 1 minute and examined with a field emission scanning elec tron microscope (JEOL 6330F). The presence of crystal was verified and crystals were identifi ed morphologically. The compositions of crystals were examined using EDS. Also SEM pictures provided image analysis of the average size distribution of calcium oxalate crysta ls formed from each experiment. The morphology of COM crystals used for AFM studies, was al so verified using the SEM method described above. Also, to confirm the hydra tion state of calcium ox alate crystals formed, energy dispersive x-ray was used.

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55 Crystal-Cell Interaction Study Preparation of COM Crystals for AFM Study COM particles used in the AFM study were gr own by homogeneous nucleation in order to obtain a single COM particle with no twinning and larger than 10 m in diameter. All glassware was acid washed. Water used in this experiment was nanopure deionized wa ter. One liter of two solutions, 10-3 M CaCl2 (CaCl2, A.C.S. Grade, 74%-78% assay, Fisher Scientific Inc.) and 10-3 K2C2O4, (K2C2O4, A.C.S. Grade, 100.8% assay, Fisher Scientific Inc.) were made in a buffer of 0.1M NaCl (NaCl, A.C.S. Grade, 99.0% assay, Fish er Scientific Inc.).The solutions were filtered through a 0.2 m filter paper (Fisherbrand, Pittsbur gh, PA). Then both solutions were stored in an incubator to equilibrate to physiological temperature, 37 C, for 24 hour. The two solutions were slowly mixed together in the incubator by dropping the K2C2O4 solution into the CaCl2 solution over the magnetic stirrer (Figure 3-4). In the incubator set at 37 C, the calcium chloride solution was transferred to a 2 liter beaker and stirred in medium speed. The potassi um oxalate solution was added to the calcium chloride solution using a titration pipette at the rate of approximately 1 l/hr. After adding all the potassium oxalate solution to the calcium chloride solution, the mixed solution was left unstirred in the incubator for 22 hours. After both solutions were completely mixed, the stirrer was turned off and the mixed unstirred solution was left in the incubator at 37 C for at least 22 hours. The crystals formed after 22 hours were collected by vacuum filtration with a 10 m filter paper (Fisher NYL Membrane 10 m CAT# R99SP04700). The filter residue s were washed thoroughly with COM saturated solution to extract the smaller size crystals. The filtered COM particles were washed

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56 with saturated COM solution to reduce dissolution of particle and then freeze-dried. The dried particles were kept at -20 C for further use. Figure 3-4 Instrument setup for mixing solution in the incubator to prepare COM crystals at 37 C. Artificial Urine Solution Preparation Artificial urine solution was prepared accordi ng to compositions shown in Table 3-3[36]. However, an artificial urine so lution used in all experiments in this study has been altered to leave out sodium citrate, calcium chloride, and sodium oxalate. These co mponent concentrations were adjusted and added later to match desire d concentrations for th e designed experiments. All the components were added to nanopure wate r in the order shown in Table 3-3. Then the solution was filtered by vacuum filtering (0.22 m Nylon, Osmonics Inc.) and stored at 4 C

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57 for up to a week. Before each AFM experiment, th e artificial urine solu tion temperature was adjusted to 37 C. The pH of the artificial urine solution was adjusted to 6.6 using sodium hydroxide or hydrochloric acid. Table 3-3 Total concentration of artificial urine displayed by the order of addition. Compound Solution Concentration (M) NaCl 0.10554 NaH2PO4 2H2O 0.03654 MgSO4 0.00385 Na2SO4 0.01695 KCl 0.06374 NH4Cl 0.03632 NH4OH 0.00062 Cell Culture Two cells lines were selected to use in this study, LLC-P K1, a proximal tubular cellular membrane derived from porcine kidney, a nd MDCK, a collecting duct cellular membrane derived from canine kidneys. Both LLC-PK1 a nd MDCK cell lines have microvilli brush border and form continuous monolayer in culture with uniform cell-to-cell contacts. The difference in structure of microvilli between the two cell li nes was discussed earlier in chapter 2. Mardin-Darby Canine Kidney (MDCK) and Porcine Kidney (LLC-PK1) cells were obtained from American Type Culture Collec tion (ATCC, CCL-34 and CL-101; Manasses, VA). Cells were maintained as conti nuously growing monolayer in culture in growth media (1:1 ratio Dulbeccos modified essential medium nutri ent mixture and F-12 (D MEM/F-12, Gibco BRL, Grand Island, NY) containing 10% newborn calf serum, 15 mM HEPES, 20 mM sodium bicarbonate, 0.5 mM sodium pyruva te, 17.5 mM glucose, streptomycin and penicillin) in 75 cm2 Falcon T-flask (12-565-52; Fisher, Norcross, GA) at 37 C in a 5% CO2 air atmosphere incubator. Under these conditions, cells achieved confluen ce. Cells were subcultured by disassociation with 0.05% tryps in and 0.25% ethylenediaminet etraacetic acid (EDTA) and

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58 seeded to 12-well plates (07-200-82; Fisher, Norcross, GA) containing Thermanox slide (NC9068818; Fisher, Norcross, GA) attached to an AFM aluminum stage. After reaching confluence, growth media was removed by acclim atization media (500 ml DMEM/F-12, 10 ml of antibiotic-antimytotic solution, 5 ml of insulin/transferring/selenium mix, 1 ml of hydrocortisone, 3.4 ml of tiiodo-L-thyronine, 1 ml of prostaglandin E1) for 8 to 12 hours. The acclimatization media were removed and re placed with artificial urine right before AFM measurements. When transf erring the cells to the AFM li quid chamber, small amount of artificial urine solution remained on the top of the cells to keep the cells from drying out and dying. Direct Force Measurement between COM Crystal and Kidney Renal Cells Using AFM An atomic force microscope was used to measure the direct force interaction between COM crystal and kidney epithe lial cell membrane. A single CO M crystal was glued (Epon R Resin 1004 F from Shell Chemical Co) to a si licon nitride AFM tip by using a tri-axial micromanipulator unit attached to an optical microscope. The fo rce measurements were obtained following the method by Ducker and Senden [63] The AFM tip with CO M crystal is shown below in Figure 3-5. An atomic force microscope (AFM) with a Nanoscope III controller form Digital Instruments, CS, was used for the force m easurements and operated by Dr. Yakov Rabinovich. Triagular cantilevers (NP-S type) from Veeco Instruments, CA, with a normal spring constant of 0.12 N/m were used. The single COM crystal att ached to the AMF tip ha d average size of 14-16 m. All the force measurements were carried out in the standard liquid chamber of the AFM. All measurements were in artificial urine solu tion or Tris buffer so lution with different concentrations of compounds in questions.

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59 To study the effect of Khella on the interac tion between COM crysta l and MDCK cells, the AFM measurement was done in artif icial urine solution (AUIS) medi um as a control experiment and in AUIS with 1mg/ml of Egyptian Khella extract. Figure 3-5 An AFM tip attached with a singl e COM crystal used fo r crystal-cell force measurement.

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60 CHAPTER 4 RESULTS AND DISCUSSION Khella Extract Active Component Characterization Several characterization techniques (see materi als and methods) were used to identify both inorganic and organic component s in the extract solution. Inorganic Component Identification An inductively coupled plasma (ICP) was used to identify single inorganic components, such as calcium, and magnesium in both Egyptia n and Turkish freeze dried extract solution. The amount of each element is shown in Table 4-1. Table 4-1 ICP results for Egyptian and Turkish extract freeze dried flakes Concentration (mg/L) Type of Extract Extract Dried-flake Mg2+ Ca2+ K+ Egyptian Khella 180 1.88 2.9 13.84 Turkish Khella 180 1.22 8.34 11.11 The ICP results show that both extracts contained magnesium, cal cium, and potassium which are reported in the literature to affect nucleation of calcium oxalate crystals. The magnesium and potassium levels in the two extr acts are relatively close, while the calcium content of the Turkish Khella extract is triple that of the in Egyptian extract. Oxalate concentration was determined usi ng the oxalate kit from trinity biotechnology. Both extracts contain relatively insignificant amount of oxalate (approx imately around 0.01mM). Organic Component Identification The organic active components in Khella extr act, such as khellin and visnagin, were identified using thin layer chromatography ( TLC) and high performance liquid chromatography (HPLC).

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61 Thin liquid chromatiography (TLC) The TLC method was used to do a quick scr eening on the organic component content of each extract. TLC showed no khell in and visnagin in the Egyptian extract while there were orange/brown spots, which match the standard khellin and visnagin, shown in the Turkish extract. High performance liquid chromatography (HPLC) HPLC experiments were done to confirm th e TLC results and also to quantify the concentration of each component in the extract. Using a pure standard of khellin and visnagin in HPLC, the peaks of khellin and visnagin appe ar at 29 minutes and 33 minutes respectively (Figure 4-1). Several concentratio ns of standard khellin and visn agin were used in the HPLC experiments to obtain the calibration curve that ca n be used to estimate the amount of khellin and visnagin in the seed extracts from area of the peak (Figure 4-2). Figure 4-1 HPLC graph of 0.5 mg/ml of pure standard khellin and visnagin

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62 y = 2.20E+07x R2 = 9.82E-01 y = 1.59E+07x R2 = 9.82E-01 0.0E+00 5.0E+06 1.0E+07 1.5E+07 2.0E+07 2.5E+07 00.20.40.60.811.2 mg/mlArea Khellin Visnagin Figure 4-2 Calibration curve for determination kh ellin and visnagin concentration from HPLC peak area. Figure 4-3 HPLC graph of Egyp tian Khella seed extract.

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63 Figure 4-4 HPLC graph of Turkish Ammi visnaga seed extract. The HPLC shows that the extract from Egyp tian Khella seeds contained no khellin or visnagin while the Turkish Ammi visnaga certified seed extract shows peaks for khellin and visnagin. This result correlates well with th e khellin and visnagin identification by TLC. The reason that Egyptian Khella seeds extrac t shows no sign of khellin and visnagin might be because of improper storage at the local herb alist which leads to the degradation of khellin and visnagin compounds in the seed. It is also possible that th e Khella seeds received from Egypt are not Ammi visnaga but a close relative species called Ammi majus [11]. As seen in Figure 4-3, the Egyptian Khella se ed extract has small p eaks at the separation time of khellin and visnagin. However, both peaks are very small. From this it can be concluded that the extract do not have any significant amount of khellin and visnagin present. The Turkish Ammi visnaga extract shows clear peaks of khellin and visnagin and matches the chromatogram

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64 of the standards. Three repe titions of the HPLC experiments were performed for the quantification of khellin and visnagin in Turkish Ammi visnaga seed extract (Figure 4-4). The amounts of khellin and visnagin estimated using the calibration curve in Turkish Ammi visnaga seed extract (1 g of dry seed/ 10 ml of wa ter) are 0.42 mg/ml and 0.18 mg/ml, respectively Even though the Egyptian extract does not show the presence of khellin or visnagin which according to the literature are the active components, it is be ing used by herbalists for the treatment of kidney stones Therefore, both seed extracts are te sted in our studies. There might be other active components that are common in both ex tracts which could be active components that effect calcium oxalate crystallization. Effect of Khella Extract on Calcium Oxalate Crystallization Effect of Khella Extract on Nuclea tion of Calcium Oxalate Crystals A nucleation study on the effect of Khella was conducted as it is the first step of crystallization. Both Khella extracts were test ed and compared with pure calcium and oxalate control solutions. The responses on nucleation m echanism tested included induction time, crystal morphology, surface energy, nucleation, and aggr egation rate approxima tion, and Gibbs free energy calculation. Light absorption measurement A UV-Vis spectrometer was used in the nuclea tion study to measure the induction time of crystals formation and to estimate nucleation, and aggregation rate. I nduction time, nucleation rate and aggregation rate can be estimated from the plot of li ght absorbance as function of time (Figure 2-6 and Figure 32). An example of the effect of Khella extract on light absorbance measurement is shown in Figure 4-5.The induc tion time at each supersaturation ratio was estimated and the values are shown in Table 4-2. The standard deviations from triplicates are also shown in Table 4-2.

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65 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 020040060080010001200140016001800Time (s)Absorbance Control 1mg/ml Egyptian extract 1 mg/ml Turkish extract Control induction time with Turkish Khella extract induction time Figure 4-5 Effect of Khella ex tract on light absorbance at initi al supersaturation of 2.0. Table 4-2 The effect of Khella extract on indu ction time of calcium oxalate nucleation from UV-Vis light absorbance measuremen t at various supersaturations. Induction time (s) SS Control with Egyptian Khe lla with Turkish Khella 2.0 376 N/A 675 2.2 128 289 252 2.4 88 188 377 2.6 74 126 85 2.8 38 78 111 The results show that both extracts prolong the induction time at ev ery supersaturation ratio, which means the system needs a longer pe riod to nucleate detectable crystals. Also the absorbance is lower which means the system form s fewer detectable crystals when compared to the baseline (control) experiment where there was no extract. From Appendix A, it can be seen that both extracts retard nucleation induction time and lower nucleation rate since the maximum increasi ng slope of absorbance is lower than for the

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66 control. The same trend applies for all supersat uration ratios tested. This observation is in contrast to the hypothesis of the effect of extract calcium concentration. Also from the light absorbance measurement, the inhibition on nucleation and aggregation can be estimated using method proposed by Hess. The percent of nucleation inhibition can be calculated by: % inhibition of nucleation = [1-(SNm/SNc)]x100 (4-1) % inhibition of aggregation = [1-(SAm/SAc)]x100 (4-2) SNm is the maximum slope of increase of absorb ance with time for the experiment with a modulator of interest. SNc is the maximum slope of increase in absorbance with time for the control experiment. SAm is the maximum slope of decrease of absorbance with time for the experiment with a modulator of interest. SAc is the maximum slope of decrease of absorbance with time for the control experiment. Using Equation 4-1, the percent nucleation inhibitory effect of Khella extract is shown in the Figure 4-6. 0 10 20 30 40 50 60 70 80 90 100 2.02.22.42.62.8 Supersaturation% Nucleation Inhibitio n Egyptian Khella Turkish Khella Figure 4-6 Nucleation inhibitory e ffect of both Khella extracts.

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67 It is clear that nucleation is greatly inhibited with for all su persaturation studies with the addition of Khella extract. At supersaturation 2.0, the light ab sorbance curve with the Egyptian Khella extract does not show an increase of slope, therefore, a 100% nucleation inhibition is assumed. Both Khella extracts show more than 50% inhibition of nucleation at every supersaturation. Thus, the additi on of both Khella extracts preven ts nucleation of calcium oxalate crystal. As the extracts delayed the absorbance maximu m, the data collected do not allow us to draw definite conclusions on their effect on aggregation of calcium oxalate crystals (see Appendix A). However, the data with supersat uration 2.8 do indicate that both extracts may retard aggregation. From the ICP measurements, it can be concluded that both herb extracts contain some level of calcium, thus the addition of Khella extract into the mixture should have increased the calcium concentration resulting in a highe r supersaturation ratio. If th e nucleation of calcium oxalate crystals is induced at a higher supersaturated solution, also le ading to a decrease in induction time, the nucleation mechanism would be based on supersaturation alone. However, the results show retardation of indu ction time and inhibition of nucleati on so it can be assumed that some constituents in both extracts act as inhibitor of nucleation. This could be done by changing surface energy, complexing free calcium or oxalate i ons in the solution, a ll of which will prevent crystal formation Calcium titration measurement A calcium titration study was done to confir m the light absorption measurements. The amount of free calcium in the so lution was detected using a calc ium electrode. The depletion of calcium concentration indicates th at free calcium ions become less available due to precipitation of calcium oxalate crystals.

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68 0 0.1 0.2 0.3 0.4 0.5 0.6 020040060080010001200140016001800Time (s)Free [Ca++] in solution (mM) Control with Egyptian Khella extract Egyptian Khella extract and oxalate only Figure 4-7 Effect of Khella extr act on titration measurement of fr ee calcium ion in solution using calcium electrode at supersaturation 2.4. The titration measurement shows that the addi tion of Khella (Egyptian) extract maintains the level of free calcium ions in the solution (Figure 4-7). The higher amount of free calcium ions initially with the addition of Khella extract is explained by the ICP resu lts that show that the Khella extract contains calcium. Therefore, some substances in the Khella extracts might compete with oxalate to bind free calcium ions which would lower the precipitation of calcium oxalate crystals and increase the induction time. Also, the titration show s that when mixing the extract with only oxalate solution, there is no calcium depletion as seen in the control. Crystal morphology studies The crystal morphology of each nucleation e xperiment was studied using both optical microscope and SEM. The SEM results show that the crystal st ructure of the control solution at every supersaturation is of only calcium oxalate monohydr ate crystals (Figure 48). Interestingly, the crystal morphology from the solution with both ex tracts contains the cal cium oxalate dihydrate form instead of COM crystals (Figures 4-9 and 4-10).

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69 Figure 4-8 SEM picture of COM crystals formed at SS 2.8. A and B are EDS intensity peaks at line A and B on COM crystals.

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70 Figure 4-9 SEM image of the calcium oxalate cr ystals formed at SS 2.8 with 1 mg/ml of Egyptian Khella extract. A) EDS intensity peak of COD crystal (line A) B) EDS intensity peaks o f calcium oxalate crystal at line B.

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71 Figure 4-10 SEM picture of calcium oxalate crystals formed at SS 2.8 with 1 mg/ml of Turkish Khella extract. A) EDS peaks at torus structure (line A). B) EDS peaks at COD structure (line B).

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72 Figure 4-11 SEM picture of calcium oxalate crystals formed at SS 2.4 with 1 mg/ml of Turkish Khella extract. The calcium oxalate trihydrat e structure was not found in any solution. However, the solution with Turkish extract has the torus structure (donut-shape) pa rticle. The torus structure of the higher supersaturations (Fi gure 4-10) has more rounded edge s than those at lower super saturations (Figure 4-11). It is also seen that the torus structure at low supersaturation (Figure 411) has a twinning structure close to the COM crystals. Therefore, it is suspected that the torus structure is the transformation structure from COM crystal. Some components in the Turkish extracts might specifically adsorb onto the CO M crystal face and inhibit growth at that surface leading to the rounded structure at higher supers aturation. The change in the calcium oxalate structure nucleated in the presence of the extrac ts is beneficial to kidney stone treatment.

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73 Wesson and Ward reported that COD struct ure showed lower adhesion surface force leading to less aggregation of crys tal and less crystal adherence to the epithelial cells [64]. Also, the torus structure might be help ful since the particle is relatively small compare to COM and COD and the smooth edge of particle might do less damage to the kidney epithelial cells and thus might be easier to pass through the nephrons The EDS results on each structure confirmed that all the crystals forms are the compone nts of calcium, oxygen and carbon which are the component of calcium oxalate structure. Other components such as potassium and chloride are also occasionally found due to th e contaminations or due to the drying effect of the salts from original solutions of calcium chloride and potassium oxalate. Surface energy and Gibbs free energy calculation The surface energy and Gibbs free energy can be calculated using the classical homogeneous nucleation theory discussed in Chap ter 2. Sohnel and Garside had stated that by plotting log (Tind) against 1/ (log SS)2 over a wide range of supersaturation a line with two different slopes will be obtained (Figure 4-13). The slope at high supersaturation ratio is for a homogeneous dominated nucleation. The slope at low supersaturation ratio is for a heterogeneous dominated nucleation. y = -4.7157x + 8.6197 0 1 2 3 4 5 6 0.60.811.2ln SSln Tind Figure 4-12 Linear relationship between ln SS and ln Tind for the control solution.

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74 Heterogeneous nucleation dominance Homogeneous nucleation dominance y = 1.5568x 2.4954 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 051015202530351/ ( lo g SS ) ^2log Tin d Figure 4-13 Induction time as a function of s upersaturation plot used for surface energy calculation. Because there are time limitations for doing expe riments at high supersaturation ratios, the supersaturation ration range of 2.0 to 2.8 wa s selected for our experiments. From the experimental data with supersaturation 2.2 to 2.8, a linear relationship between natural log of supersaturation and natural log of induction time is obtained (Figure 4-12). Then this linear equation is used to extrapolate the induction tim e over a wide range of s upersaturation [65, 66]. The slope of a homogeneous dominated nucleation straight line is used to calculate surface energy since our experiments are based on homogeneous nucleation. SEM pictures show that with the addition of Khella extract, the calcium oxalate crystals have a primary form of calcium oxalate dihydr ate. Therefore, when calculating the surface energy for the experiment with the addition of Kh ella extract, we need to use the constants for COD. Also the amount of calcium ions in the extract is considered to have an effect in changing supersaturation ratios of the system. The new s upersaturation ratios are calculated. It is found

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75 that Khella extract lowers supersaturation despit e the fact that the extract itself increases the calcium concentration of the system (Tables 4-3 and 4-4). These new supersaturation ratios are used in the calculation of surface energy of the calcium oxalate crystals that formed in the pres ence of Khella extract. The surface energy values obtained are shown in Table 4-5. Table 4-3 New supersaturation ratio calculation of the system with Egyptian extract. Starting Concentration (mM) From Egyptian Khella Extract (mM) total (mM) SS [Ca2+] [Ox2-] [Ca2+] [Ox2-] [Ca2+] [Ox2-] New SS using COD constants 2.0 0.430 0.430 0.0724 0.01 0.502 0.440 1.65 2.2 0.473 0.473 0.0724 0.01 0.545 0.483 1.80 2.4 0.516 0.516 0.0724 0.01 0.588 0.526 1.95 2.6 0.559 0.559 0.0724 0.01 0.631 0.569 2.10 2.8 0.602 0.602 0.0724 0.01 0.674 0.612 2.25 Table 4-4 New supersaturation ratio calculation of the system with Turkish extract. Starting Concentration (mM) From Turkish Khella Extract (mM) total (mM) SS [Ca2+] [Ox2-] [Ca2+] [Ox2-] [Ca2+] [Ox2-] New SS Using COD constants 2.0 0.430 0.430 0.208 0.01 0.638 0.440 1.86 2.2 0.473 0.473 0.208 0.01 0.681 0.483 2.01 2.4 0.516 0.516 0.208 0.01 0.724 0.526 2.17 2.6 0.559 0.559 0.208 0.01 0.767 0.569 2.32 2.8 0.602 0.602 0.208 0.01 0.810 0.612 2.47 Table 4-5 Effect of Khella ex tract on surface energy of calcium oxalate crystals (*indicating using COD constants) Surface Energy (mJ/m2) Control 19.13.19 with Turkish Khella Extract 18.75.78* with Egyptian Khella Extract 16.35.13* The surface energy calculated for COM in de ionized water (control solution) is 19.13 mJ/m2. The calculated surface energies in aqueous solution using constant composition methods by other investigators are approximately 13-29 mJ/m2 [67, 68]. When compare our surface

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76 energy with others, the values are relatively clos e, so the surface energy determination from the induction period in our study is reasonable. The calculated surface energies are lower for th e calcium oxalate crys tal nucleated in the system with addition of Khella extract. This impl ies that calcium oxalate crystals in solution with Khella extract should be easier to form, which is in contrast to the results from the induction times. The surface energy values for the system with Khella were cal culated using the COD constants since it is the main crystal morphology shown in SEM pictures. However, considering the standard deviation, the surface energy diffe rences can be considered close together. Haselhuhn et al also compared the surface energy of COM and COD crystals formed at 50 C. They showed that COD crystal has slightly lower value than that of COM. However, their range of surface energies, 90 mJ/m2 for COM and 70 mJ/m2 for COD, are much hi gher than our values since the experiments are done in different temper ature, but, it shows th e same trends. The COD crystal is known to be a precursor for the COM crystals in biological systems. Therefore, COD crystals are believed to form first then transf ormed to COM crystals, which should be able to explain the lower surface energies in the presence of Khella extract. However, this is in contrast with the phase stability of calcium oxalate crys tals. COM is the most stable form of crystal therefore the surface energy of nuclei should be the lowest of all other forms. The surface energy is used for calculation of Gibbs free energy of formation. The calculation is based on the homogeneous nucleat ion assumption, where the value is the summation of a volume term and a surface term as mentioned previously in Chapter 2. 2 34 3 4 r G r r Gv N (4-1) where GN is Gibbs free energy of formation/nucleation, Gv is the change in volume free energy, r is radius of nuclei, and is surface energy.

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77 As mentioned before, the critic al free energy barrier of formation is the point that the energy from the volume term overcomes the surface te rm. Therefore, to calc ulate the critical free energy barrier of formation, Gcrit, the derivative of Gibbs free en ergy of formation with respect to the radius of nuclei, d( GN)/dr is set to equal zero and set r to equal critical radius of nuclei, rcrit (Equation 4-2 and 4-3). 0 8 4 ) (2 r G r dr G dv N (4-2) crit v critr G r 8 42 (4-3) By rearranging the terms, the change of volume free energy, Gv, is equal to crit vr G 2 (4-4 Also, Gcrit can be calculated from GN at r equals to rcrit as shown below. ) (crit N critr G G (4-5) Substituting rcrit to equation 2 34 3 4crit v crit crit Nr G r r G (4-6) Then substituting Gv from Equation 4-4. 2 2 33 4 4 ) 2 ( 3 4crit crit crit crit crit Nr r r r r G (4-7) 23 4crit critr G (4-8) Also, by rearranging Equation 4-4, the rcrit value can be written in Gv term. v critG r 2 (4-9) Therefore, by substituting rcrit from Equation 4-8 and rearrang ing the terms, we obtain the equation of change in volume free energy, Gv, in the term of the cr itical free energy barrier (Equation 4-10). 2 1 33 16 crit vG G (4-10)

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78 Thus, substituting the Gv value from Equation 4-10 to Equation 4-1, we get the equation of Gibbs free energy as the f unction of nuclei radius and Gcrit 2 2 1 3 34 3 16 3 4 r G r G r Gcrit crit N (4-11) From the Gibbs-Thompson equation, rcrit is obtained in terms of molar volume (VM,) surface energy ( ), supersaturation ratio (S), gas cons tant (R), and temperature (T) [69]. crit M wRTr V r RT M S 2 2 ln (4-12) S RT V rM critln 2 (4-13) Therefore, substituting rcrit from Equation 4-13 to Equation 4-8, we get 2 2 3 2 2) (ln ) ( 3 16 ln 2 3 4 S RT V S RT V GM M crit (4-14) From these derivations, the Gibb free energy of formation can be calculated using the surface energy and supersaturation ratios. Figure 4-14 shows the decrease in free energy ba rrier and critical radius of nuclei with the increase of supersaturation ratio. This result confirms the faster induction time of higher supersaturation of calcium oxala te. At high supersaturation, th e system requires lower energy and smaller nuclei to precipitate stable crystals. The critical free energy barrier and the critical radius of nuclei are lower at hi gher supersaturation, leading to fa ster nucleation rate of calcium oxalate crystals. The effect of Khella extract on the change in Gibbs free energy of formation was also studied (Figure 4-15 and Appe ndix B). However, for the systems with extract, the supersaturation is lower (Tables 4-3 and 4-4). It was decided to plot, the change in Gibbs free energy against the initial supers aturation of calcium and oxalate concentration (based on the amount added)

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79 -5E-21 0 5E-21 1E-20 1.5E-20 00.10.20.30.40.50.60.7Radius (nm)Change in Gibbs Free Energy (J)S = 2.0 S = 2.8 S = 2.6 S = 2.4 S = 2.2 Figure 4-14 Change in Gibbs free energy of form ation with respect to supersaturation ratios From Appendix B, the Gibbs free energy barrier and critical radius of nuclei increases with the addition of Egyptian Khella ex tract or Turkish Khella extract at every initial supersaturation of calcium and oxalate solution. Both extracts shift the critical nuclei radius to larger values and also increase the free energy barriers of formati on. Therefore, the system needs larger nuclei and higher energy to overcome the barrier in order to precipitate stable crystals. These results explain the longer induction time effect of both Khe lla herbal extracts. Since COD crystals are less stable than the COM crystals, the higher critical energies barrier and higher critical radius of nuclei explains th e observation of COD crystals when Khella extract is added. The phase transformation of COD to COM occurs because the Gibbs free energy of COM crystals is lower as shown in the control solution. When the system reaches

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80 equilibrium, it should go to the lower energy stat e of COM. However, the crystals observed after 30 minutes of light absorbance measurement remain ed in dihydrate structures for both Khella extracts. Therefore, some substances or com ponents in the Khella ex tract might prevent the phase transformation of COD to COM in the systems. -1E-20 -5E-21 0 5E-21 1E-20 1.5E-20 2E-20 2.5E-20 0.00.10.20.30.40.50.60.70.80.91.0Radius (nm)Change in Gibbs free energy of formation (J) Control with Egyptian Khella extract with Turkish Khella extract Figure 4-15 Effect of Khella extract on GN at initial supersat uration of SS 2.0 The critical energy barrier is also plo tted with respect to each systems actual supersaturation in Figure 4-16. Th e plot shows that both extracts have no significant change in critical free energy barriers at ev ery supersaturation. Therefore, it can be concluded that the longer induction time effect of Kh ella extract is based on the lowe r supersaturation of the system by forming and preserving the calc ium oxalate dihydrate crystals.

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81 Figure 4-16 Effect of Khella ex tracts on the change in critical free energy barrier at different supersaturation ratios. Cell-retention of calcium oxalate crystal The interaction force between cell lines, LLC-PK1 and MDCK and COM crystal was studied using AFM. It was found in earlier studies that there is no attraction force between LLCPK1 cells and a COM crystal at tached to the tip [19]; only repulsion was found in both long range and short range for all c onditions of LLC-PK1 cells and all solutions used in the experiment. The no adhesion force with the LLC-PK1 cells agrees with the fact that there is a lack of crystal deposition in the proximal tubular section (representati on of LLC-PK1 cell line). However, adhesion force is found for the MDCK epithelial cell and a CO M crystal on the AFM tip in artificial urine solution medium. This ma y be due to the different brush border member structure and biochemistry of the cells. Figure 2-4 shows the differences in cell brush border membrane structure. The cell membrane structur e of the proximal tubul ar (LLC-PK1) section

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82 has long and close-packed microvilli, while the cell membrane structure of the collecting duct (MDCK) section has short and smooth microvilli. Also, crystal adhesion to the cells depends on the structure of the brush border membrane, the differences in protein composition of the cells and the flow rate of urine in each section. Therefore, this stu dy is focused more on the adhesion force between COM crystal and MDCK cells. -0.5 0 0.5 1 1.5 2 05001000150020002500 Indentation Distance (nm)F (nN) Control Khella extract Figure 4-17 Effect of Khella extract on COM crystals and MDCK interaction force by AFM. To measure the adhesion force between CO M crystal and MDCK cells, the force was measured during the retraction of the AFM cantil ever out of the surface. Upon approaching the COM crystal on the cantilever tip to the MDCK cells, it causes a pus h to the cell itself and the repulsion force is caused by the elasticity of the cell. When slowly removing the cantilever from the cells, the repulsion force (positive force) due to the cell elasticity decreases until the curve crosses the x-axis which is the point when the cel l returns to its initial stage. Then the negative force is measured if there is adhesion between the COM crystals and kidney epithelial cells.

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83 Figure 4-17 shows no negative inte raction force when the Khella extract is present. The error bars indicate standard deviations at the point of maximum a dhesion. Some raw AFM measurements are shown in Appendix C. The abse nce of negative interaction force result implies that there is no adhesion force between COM and MDCK cells. Ther efore, Khella extract might have components that prevent the COM crystals from sticking to the MDCK cells. It is reported that the kidney epithelial cells have the affinity to attract COM crystals due to electrostatic attraction between the calcium-rich phase of calcium oxalate crystals and the anionic charge of the phospholipids of the epithelial cell membrane. Khellin and visnagin was reported to have antispasmoic action to smooth muscle cells. Both khellin and visnagin have the calcium channel blocking which prevents calcium ions from permeating throug h the calcium channel. The calcium ions releasing action of the calcium channel is the signal sent to the smooth muscle cell to retract. When the channel is blocked, the sign al from the calcium channel can not be sent so the cell is relaxed. Khella extract, might have the same effect on MDCK cells so MDCK cell interaction to calcium is limited, hence the MD CK cell is relaxed and does not exert adhesion force to COM crystals. Effect of Khella Extract Components on Calcium Oxalate Crystallization The previous section shows that the Khella extract has positive resu lts in preventing the kidney stone formation. Addition of Khella extrac t reduces the supersatura tion of the system and lengthens the induction time. Also, the majority of calcium oxalate crystals formed in the presence of Khella extract is in dihydrate fo rm instead of monohydrate form. The Turkish Khella extract also has the torus struct ure of calcium oxalate crystals which is suspected to be slow growing COM. This result might be due to specif ic site absorption of some components of the extract. Therefore, the Khella extract component s, khellin, visnagin, calcium, and magnesium, needs to be investigated on cal cium oxalate crystallization.

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84 Effect of Khellin and Visnagin on Calcium Oxalate Crystallization Khellin and visnagin are the organic component s of the Khella extract identified from the HPLC as well as in the literature. Khellin and vi snagin are believed to act as active constituents that effect curing or preventing the reoccurrence of kidney stone. Nucleation studies Pure khellin and visnagin were tested usi ng a range of concentration according to the literature. Literature states that khella had a pproximately 0.3-1.2 wt% of khellin and 0.05-0.3% of visnagin [11]. A statistical de sign of experiments is used to de termine the significant effects of both components. Khellin and visnagin are th e factors of the desi gn. Induction time and nucleation inhibition per cent are the responses. Table 4-6 Three level composites design experimen t: the effect of khellin and visnagin on light absorbance measurement Concentration (mg/ml) Std # Run # Khellin Visnagin Induction time (s) % Nucleation Inhibition 1 5 0 0 126 0 2 13 0.015 0 150 27 3 9 0.03 0 140 37 4 1 0 0.0025 220 39 5 12 0.015 0.0025 120 -1 6 7 0.03 0.0025 137 21 7 4 0 0.005 140 5 8 6 0.015 0.005 139 15 9 3 0.03 0.005 135 -28 10 10 0.015 0.0025 120 3 11 8 0.015 0.0025 160 17 12 11 0.015 0.0025 130 -3 13 2 0.015 0.0025 150 -3 Three levels were selected for each khellin and visnagin with zero is the lowest concentration and the highest concentration 0.03 mg/ml for khellin and 0.005 mg/ml for visnagin. Three level composites design was sel ected for this study. Supersaturation 2.4 by adding calcium and oxalate soluti on was chosen for this experime nt. Table 4-6 shows the design

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85 concentration of khellin and visnagin and the re sponces, induction time and nucleation inhibition percent, for each run. % Nucleation InhibitionKhellin (mg/ml)Visnagin (mg/ml) 0.0000 0.0075 0.0150 0.0225 0.0300 0.0000 0.0013 0.0025 0.0037 0.0050 -9 1 1 11 21 30 5 5 5 5 5 % Nucleation InhibitionKhellin (mg/ml)Visnagin (mg/ml) 0.0000 0.0075 0.0150 0.0225 0.0300 0.0000 0.0013 0.0025 0.0037 0.0050 -9 1 1 11 21 30 5 5 5 5 5 Figure 4-18 Contour plot from statistical de sign on % nucleation inhibition of khellin and visnagin. Using ANOVA to analyze the data set, no st atistical significance model could fit the change of induction time for khellin and visnagin. Therefore, both khellin and visnagin have no significant effect in changing the induction tim e on nucleation of calcium oxalate crystals. The nucleation inhibition effect is analyzed using 2FI model, where khellin, visnagin, and the interaction between bot h of the components are studied. Th e standard run # 4 is blocked due to the lack of fit. ANOVA shows that visnagin and the two interaction terms are significant model terms. The contour plot shows the intera ction effect of khellin and visnagin on the nucleation inhibition of cal cium oxalate crystals. The contour plot shows that th e highest nucleation i nhibition effect is where the system has high khellin and low visnagin where nucleation inhibition has a value approximately 30%. At high khellin and high visnagin, the nucleation in hibition is lowest, -9 %. However, when comparing this result to the effect of Khella extract on nucleation inhibition, we see that the

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86 Khella extract has much higher inhibitory effect on nucleation. Therefore, khellin and visnagin are not the main component that is involved in the nucleation inhibi tory effect of Khella extract. This is of course supported by th e absence of appreciable amount of khellin and visnagin in the Egyptian Khella used in this study. Crystal morphology studies After each light absorbance measurement, the crystals were collected and prepared for SEM in the same manner as done previously. Fi gure 4-19 shows the SEM and EDS analysis of the crystals collect ed after run #11. Figure 4-19 shows the SEM pictures of calcium oxalate crystal with the effect of khellin and visnagin. It is clear that crystals formed were all calciu m oxalate monohydrate (COM). Some of the potassium chloride salts crystals are also found due to the contamination mentioned before. These results show that khellin and visn agin do not have any effect on changing crystal morphology from COM to COD or the torus structure. Figure 4-19 SEM pictures of calcium oxalate crystals formed at SS 2.4 with 0.015 mg/ml Khellin and 0.0025 mg/ml visnagin. A) EDS intensity peaks of COM crystal (line A). B) EDS intensity peaks of KCl crystal (line B).

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87 Effect of Calcium and Magnesium on Calcium Oxalate Crystallization Calcium and magnesium are also known as important factors of calcium oxalate crystallization. The ICP result shows that Khella extract contains both of these ions. Therefore, the effect of calcium and magnesium is investigated in our studies. Nucleation studies The effect of magnesium was investigated using light absorbance measurements with results shown in Appendix F. The induction time wa s estimated and is shown in Table 4-7. Also the inhibitory effect of magne sium on calcium oxalate crystal nucleation was estimated (Figure 4-20). Table 4-7 Effect of 0.1mM Mg2+ on induction time of calcium oxalate nucleation Induction time (s) SS Control with 0.1 mM Mg2+ 2 376 173 2.2 128 140 2.6 74 60 2.8 38 45 Table 4-7 shows that magnesium does not ha ve significant effect on induction time retardation. From the induction time obtained, th e surface energy of nuclei was calculated to be 18.5 .67 mJ/m2, which is relatively close to the surface energy of the control as shown previously. COM constants were used for the surface energy calculation because from the SEM picture the crystal formed has mainly COM st ructure (Figure 4-21). The SEM picture clearly shows that the change in crystal morphology seen with the presence of Khella extract does not depend on magnesium level in the extract alone. The inhibition of calcium oxalate crystal nu cleation estimated from light absorbance measurements was also calculated. Magnesium shows inhibitory effect on calcium oxalate nucleation (Figure 4-20). At supe rsaturation 2.0, the percent nucl eation inhibition has a negative value, which means it promotes nucleation. But this might be due to experimental error given the

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88 error bar shown. Nevertheless, the percent of nuc leation inhibition from magnesium is less than what we get from Khella extract. Thus it can be concluded that magnesium concentration in the extract does not have a significan t contribution on the inhibiti on effect of calcium oxalate crystallization of the Khella extract. -100 -80 -60 -40 -20 0 20 40 60 80 100 2.02.22.62.8Supersaturation% Nucleation inhibition with 0.1 mM Magnesium with Egyptian Khella extract with Turkish Khella extract Figure 4-20 Inhibitory eff ect of magnesium on calcium oxalate crystal nucleation. J. Jeon and H. El-Shall investigated the e ffect of high calcium ratio, hypercalciuria, previously [39]. It was found that hypercalcuri a, has faster induction time, as well as the hyperoxaluria, high oxalate, because th e supersaturation is increased. Also high calcium ratio has a structural modifi cation effect on the calcium oxalate crystals nucleated. The crystals formed with high level of calcium ratios had rounder edges as seen in Figure 4-22. This structure is closer to th e bi-pyramid structure of COD than the COM monoclinic structure. However, there was no defined COD structure observed in this high calcium concentration in contrast to wh at was observed with Khella extracts.

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89 Figure 4-21 SEM picture of calcium oxalate crystals formed at SS 2.6 with the addition of 0.1 mM magnesium. Figure 4-22 Calcium oxalate crys tals in high calcium concentration (taken from Jeons thesis) [39].

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90 Several additives are suggested in the lit erature to induce the COD crystals in in vitro crystallization studies. High cal cium to oxalate ratio was claimed to be one of preferable conditions for COD [70]. Therefore the struct ure observed in Figure 4-22 might be calcium oxalate crystals during the transformation of le ss stable COD to a stable COM structure. To sustain the COD crystal, the system should have other substances that can stabilize the COD phase. Magnesium was suggested to play this stabilizing role for th e COD structure [71]. Therefore, the COD crystals formed with the presence of Khella extr act might involve the addition of calcium and magnesium from Khella extract. However, it may be an interaction effect of both calci um and magnesium. Cell retention studies Lieske et al. investigated the effect of various concentrations of calcium and magnesium in Tris Buffer Saline (TBS) solution on the 14C COM crystals attachment on MDCK cells. Both magnesium and calcium were found to have higher attachment of COM crystals to MDCK cells, hence higher adhesion [53]. AFM measurements in this study were done re peating the conditions of Leiske et al. There is a good correlation between the present AF M results and the results from Leiske et al (Figure 4-23). Both studies show a critical concentration for both Ca2+ and Mg2+ ions where the maximum adhesion forces appear. Also it was found that the solution with Ca2+ ions had much larger adhesion fo rce than the solution with Mg2+ ions (Table 4-8) The AFM results show that the adhesion fo rce first increases then decreases with increasing calcium and magnesium concentration, but is not eliminated. Recall that the AFM study on Khella extract showed no adhesion force at all. Therefore, the presence of calcium and magnesium in Khella extract has no significant cont ribution on the absen ce of adhesion force between COM crystal and MDCK cells.

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91 From these studies, it is show n that all the Khella extrac t components studied so far, khellin, visnagin, calcium, and magnesium, do not contribute to the calcium oxalate crystallization inhibitio n of the extract. Only magnesium s hows minor retardation of induction time and inhibitory effect on calcium oxalate nucleation. None of the components shows the crystal structure altering from CO M to COD or the torus structure. Also, adhesion force is shown with calcium and magnesium, while Khella ex tract prevents adhesion between COM and MDCK cells. Therefore, there should be some other compone nts in Khella extract that contribute to the inhibitory effect of calcium oxalate crystallization and cell-retention. Table 4-8 Adhesion force between MD CK cells and COM crystal in Ca2+ and Mg2+ solutions. Concentration of ions (mM) Fad for Ca2+ (nN) Fad for Mg2+ (nN) 0 0.12.01 0.12.01 25 0.20.02 50 0.38.04 0.12.01 100 0.51.05 0.20.02 200 0.27.03 0.06.01 400 0.26.03 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 050100150200250300350400450500[Ca, Mg] (mM)Fad (nN); CPMx14 1 2 3 4 Figure 4-23 Effect of Ca2+ and Mg2+ on adhesion force between CO M crystal and MDCK cells. Line 1 and 2 are effects of Ca2+ and Mg2+ respectively on adhesion force measured by AFM. Line 3 and 4 are effects of Ca2+ and Mg2+ respectively on adhesion force measured by Lieske.

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92 Effect of Other Urinary Species on Calcium Oxalate Crystallization Other urinary species such as citrate, oxala te, urinary proteins, and cellular membrane debris were also studied on thei r effect on crystallization of calcium oxalate. Some species are known to be an inhibitor of kidney stone formation, such as citrate and al bumin proteins. Others, oxalate and cellular membrane de bris, are know as promoters. Effect of Citrate on Calc ium Oxalate Crystallization Citrate is a known inhibitor of calcium oxalate crystal nucleation due to the anionic charge of citrate structure. It has been studied thoroughly on its inhibition of calcium oxalate crystallization [7, 30, 38, 47, 70, 72]. Citrate was investigated here to compare its effects to those of the Khella extracts. Nucleation studies The UV-Vis absorbance graph confirms that citrate prevents nucleation by increasing induction time at every supersaturation ratio (T able 4-9, Appendix G, and Figure 4-24). An increase in induction time with th e presence of citrate is due to the change in supersaturation ratio. When citrate is introduced to the system, the supersaturation ratio decreases because of the formation of a soluble calcium citrate complex as discussed before in chapter 2. Such a complex makes the calcium ions in the system less availa ble to interact with ox alate and form calcium oxalate crystal. Also, the percent nucleation inhi bition of citrate can be estimat ed from the light absorbance measurements. It was found that citrate also inhibited the nucleation rate. However, when compared to the Khella extract, citrate has less nucleation inhibition effect as (Figure 4-25). Also at supersaturation 2.8, citrate lose s its nucleation i nhibitory effect. The result from the SEM picture (Figure 4-26) shows that the calcium oxalate crystals formed in the presence of citrate have a flatter and less sharp edges struct ure of calcium oxalate

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93 monohydrate. Citrate is known to specifically bind on the surface of COM crystasl to minimize configuration energy. -0.005 0 0.005 0.01 0.015 0.02 0.025 020040060080010001200140016001800Time (s)Absorbance Control with 0.1mM Citrate Control induction time with 0.1mM Citrate induction time Figure 4-24 Effect of citrat e on light absorbance measurem ent at supersaturation 2.0 Table 4-9 The induction time estimation from li ght absorbance measurement with the presence of citrate. Induction time (s) SS Control with Citrate 2.0 376 425 2.2 128 290 2.6 74 131 2.8 38 83

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94 -100 -80 -60 -40 -20 0 20 40 60 80 100 2.02.22.62.8Supersaturation% Nucleation Inhibition with 0.1 mM citrate with Egyptian Khella extract with Turkish Khella extract Figure 4-25 Inhibitory effect of 0.1mM citrate on calcium oxalate nucleation. Figure 4-26 The effect of citrate on calc ium oxalate monohydrate crystal structure.

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95 Figure 4-27 EDS spectrometer of a single calcium oxalate crystal forming with citrate. The EDS spectrometry on a single crystal was performed. The result shows the peak of calcium, carbon and oxygen, which confirmed that th ese crystals were calcium oxalate. The gold peaks are also shown due to th e gold coating of the sample. Qiu et al. reported that citrat e preferable binds to a (-101) step more than a (010) face, which causes the calcium oxalate monohydrate crystal to have sm oother and rounder edges [72]. However, the calcium oxalate m onohydrate structure modification fr om citrate does not show the torus structure that was seen with Turkish Khella. Some substances present in Khella extract might act as citrate does on the crystal morphology. Some co mponents with anionic charges interact with calcium and also specifically bind to the calcium rich surface. This specific binding inhibits the growth of specifi c faces and induces the smooth a nd rounded edges like the torus structure. The calculated surface energy of the system with the addition of 0.1 mM citrate is 19.5.2 mJ/m2. When compared to the surface energy of th e control system calculated earlier which is 19.13.19 mJ/m2, it can be concluded that citrate does not have appreciable effect in changing the surface energy of nuclei, even though it changes the morphology. The critical free energy barrier at each supersaturation is calcul ated and plotted as seen in Figure 4-28. It is seen that citrat e does not change the critical fr ee energy barrier of the system at

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96 every supersaturation. Therefore, it can be c oncluded that even though ci trate increases induction time and changes crystal morphol ogy, it does not change the free energy of the system. So the crystal nucleates should still be COM in structur e. The change in crys tal morphology by citrate should be due to slower crystal growth at specific sites of CO M crystals as discussed before. 0 1E-20 2E-20 3E-20 4E-20 5E-20 6E-20 7E-20 11.522.533.5SupersaturationChange in Critical free energy barrier Control with 0.1mM citrate Figure 4-28 Effect of citrate on chan ge in critical free energy barrier. Cell-retention studies Besides the effect of citrate on nucleation of calcium oxalate crystals, cell-retention was also studied. Figure 4-29 show s the plot of interaction for ce between COM cr ystal and MDCK cells as a function of indentation. It is seen th at the presence of citrat e in AUIS solution medium reduces the adhesion forces between COM and MD CK cells. The adhesion force in the control (AUIS) solution is approximately equal to -0 .4 nN and equal to -0.25 nM in the AUIS containing 0.1 mM citrate soluti on. The differences in indenta tion distance at maximum adhesion

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97 force depend on the thickness of the cells at the measuring point where the COM crystal on cantilever tip touches the cells. -0.5 0 0.5 1 1.5 2 05001000150020002500Indentation Distance (nm)F (nN) Control with 0.1 mM of Citrate Figure 4-29 The interaction force measuremen t between COM crystal and MDCK cells in control (AUIS) and with 0.1mM of citrate in AUIS solution. Effect of Cellular Membrane Debris on Calcium Oxalate Crystallization When the renal epithelial cells are injured, the degradation of epithelial cells occurs. The membrane debris in the urine is believed to have a role in kidney stone formation because of its presence in the organic matrix of the kidney stone. Debris from MDCK and LLC-PK1 kidney ep ithelial cell membrane was added to the calcium and oxalate solution at va rious supersaturation ratios. The data showed that the debris

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98 from both cell lines enhanced the nucleation of calcium oxalate crystal by decreasing induction time at low supersaturation (Figure 4-30 and Table 4-10). -0.002 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0.02 050100150200250300350400Time (s)A Control with MDCK cell debris with LLC-PK1 cell debris With MDCK With LLC-PK1 Control -0.002 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0.02 050100150200250300350400Time (s)A Control with MDCK cell debris with LLC-PK1 cell debris -0.002 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0.02 050100150200250300350400Time (s)A Control with MDCK cell debris with LLC-PK1 cell debris With MDCK With LLC-PK1 Control Figure 4-30 Effect of cellular membrane de bris (MDCK and LLC-PK1) on light absorbance measurement at SS 2.0 Table 4-10 Effect of cellular me mbrane debris on the induction time at various supersaturation By providing nucleation sites, cellular membrane debris increases the nucleation rate. Therefore, the presence of cell membrane debris reduces a free energy barrier for nucleation. However, when considering that the standa rd deviation on induction time ranges from 10 Induction time (s) SS Control LLC-PK1 MDCK 2 163 140 100 2.2 90 90 90 2.4 42 65 60 2.8 31 40 35 3.0 25 20 20 3.2 18 15 11

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99 seconds to 60 seconds in previous experiments, it can not be conclude d that the presence of cellular membrane debris significantly changes it. -200 -150 -100 -50 0 50 100 2.02.22.42.83.03.2supersaturation% nucleation inhibition LLC-PK1 MDCK Figure 4-31 Effect of cellular membrane debris on nucleation rate inhibition. The effect of the cellular debris on calcium oxa late nucleation rate is also investigated (Figure 4-31). It shows that the presence of both LLC-PK1 and MDCK cell debris, promotes nucleation of the calcium oxalate crystal. The ne gative value of percent of nucleation inhibition implies that the system has faster nucleation ra te due to the higher maximum increased slope of light absorbance with time. Despite, the small change s in induction period, it is clear that cellular membrane debris induces nucleation by increasi ng the nucleation rate. The positive percent of nucleation inhibition at supersat uration 2.8 might be due to ex perimental errors at that supersaturation.

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100 Also, from Figure 4-31, it is seen that th e cellular debris from MDCK cell has higher inhibitory effect in most of supersaturations Therefore, the MDCK cells might have higher affinity to interact with calcium oxalate crystals than LLC-PK1 cells, something consistent with the fact that kidney stones are found more at the collecting duct section (represented by MDCK cells) than in other sections. The surface energy and Gibbs free energy with the presence cellular membrane debris are calculated and the resu lts are shown below. Table 4-11 The effect of cellular memb rane debris on surface energy of nuclei Surface Energy (mJ/m2) Control 19.28 MDCK 20.19 LLC-PK1 19.65 -1E-21 1E-21 3E-21 5E-21 7E-21 9E-21 1.1E-20 1.3E-20 1.5E-20 00.10.20.30.40.50.60.7Time (s)Change in Gibbs free energy of formation (J) Control with LLC-PK1 cell debris with MDCK cell debris Figure 4-32 Effect of cellular me mbrane debris on change in Gi bbs free energy of formation at SS 2.2

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101 Table 4-11 shows no significant change in su rface energy with the presence of cellular membrane debris. No change in surface energy im plies that the cellular membrane debris does not alter the calcium oxalate crystal struct ure or bind with the crystal surface. 0 2E-21 4E-21 6E-21 8E-21 1E-20 1.2E-20 1.4E-20 1.6E-20 1.8E-20 1.822.22.42.62.83SSChange in critical free energy barrier (J) Control with MDCK cell debris with LLC-PK1 cell debris Figure 4-33 Change in critical free energy barrie r as a function of supersaturation with the presence of cellular membrane debris It is shown in Figures 4-32 a nd 4-33 that the Gibbs free energy of formation and a critical free energy barrier with the presen ce of both cellular membranes is slightly higher than for the control. The critical radius of nuclei remains relatively close for all conditions. These results show that the presence of cell de bris does not change the free en ergy of formation which is in contrast to the faster nucleat ion rate found previously. Howeve r, all the surface energy, Gibbs free energy of formation, and critical free ener gy barrier values are calculated based on the homogeneous nucleation assumption. As disc ussed previously, the presence of cellular membrane provides the physical site for nucleation to occur. Ther efore the system with cellular

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102 debris is subjected to heterogeneous nucleati on. So the assumption of homogeneous nucleation with a system that contains cell debris is not valid. Effect of Urinary Proteins on Calcium Oxalate Crystallization Proteins are another important urinary species that may effect crystallization of calcium oxalate.. Albumin was selected to be representative of urinary proteins in this study. Albumin is the most common and most abundant protein in the biological system. Also albumin is found in the organic matrix of kidney stone s; therefore, it should be invol ved in the formation mechanism of kidney stones. Nucleation studies The light absorbance measurements were perfor med in the mixture of calcium and oxalate solution at various supersatura tion ratios. To study the eff ect of urinary proteins, the concentration of 15 g/ml of albumin was se lected. The induction time estimation from light absorbance is shown in Table 4-12. -0.004 -0.002 0 0.002 0.004 0.006 0.008 0.01 0.012 020040060080010001200140016001800Time (s)Absorbance Control with Albumin Control induction time with Albumin induction time Figure 4-34 Light absorbance measurement with th e effect of albumin at supersaturation 2.0

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103 Table 4-12 Effect of albumin protein on induction time at various supersaturations Induction time (s) SS Control With Albumin (15 g/ml) 2 172 177 2.2 105 135 2.4 56 90 2.8 31 40 The presence albumin slightly prolongs i nduction time at every supersaturation when compared with the control experiments. Howeve r, this retardation of induction time is not significant when considering the standard deviat ion. The calculation of surface energy and Gibbs free energy barriers using homogene ous nucleation can not be assume d in this case. The addition of albumin in the solution provi des the foreign body to the syst em; therefore, our nucleation system with albumin is heterogeneous nucleati on. As shown in the re sult of nucleation study with the presence of cellular membrane debris, the homogenous assumption is not valid with the heterogeneous nucleation. -100 -80 -60 -40 -20 0 20 40 60 80 100 2.02.22.42.8Supersaturation% Nucleation Inhibition with albumin with Egyptian Khella extract with Turkish Khella extract Figure 4-35 Effect of albumin on percent inhibition of calcium oxalate nucleation.

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104 The change of slope in light absorbance m easurement is noticed with the addition of albumin protein. The inhibition of albumin protein on calcium oxa late crystals nucleation was estimated using the same method as before. In addition of nucleation inhibition effect of albumin, the effect on calcium oxalate aggrega tion was also noticed. Unlike all other studies mentioned earlier in this thesis, these experiment sets with albumin show significant decreases in slope after the absorbance reaches maximum valu e (see Appendix J). The i nhibitory effect of albumin on aggregation can be es timated using Equation 4-2. The inhibitory effects of albumin on nucleation and aggregation are shown in Figures 4-35 and 4-36. -250 -200 -150 -100 -50 0 2.02.22.42.8Supersaturation% Aggregation Inhibition Figure 4-36 Inhibitory effect of albumin on calcium oxalate crystals aggregation rate. Figure 4-35 shows that albumin promotes nucle ation at low supersat uration and inhibits nucleation at higher supersaturation. Comparing to the previous results, from Khella extract that have nucleation inhibitory effect in the range of 70% or more, th e inhibition effect of albumin protein on nucleation is small.

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105 It is clear that albumin promotes aggregat ion of calcium oxalate crystals at every supersaturation. Albumin might enhance the aggregation of cr ystals due to steric attraction by forming polymer bridging between crystals. Our resu lts are in contrast with other previous study which claimed that albumin ha s a dispersive effect between calcium oxalate crystals by promoting nucleation of smaller calcium oxalate crystals [73]. Kulaksizoglu et al. also s uggested the promotion of calci um oxalate nucleation as the concentration of albumin increases [74]. Atmani et al found that albumin is present in the matrix of all type of stones [29, 75]. The presence of al bumin in the stone matrix indicates that albumin should be involved in the calcium oxalate crystals aggregation mechanism. Albumin has anionic molecular structure which has high affinity to calcium [34] whic h could explain the retardation effect of induction time. Therefore, it is suggested that albumin mi ght bind with the calcium rich phase of calcium oxalate crystals [76]. Since our experiments were done in an unstirred system, the decreased slope of light absorbance is dependent on the crystal settling rate. The settling rate depends on se veral factors such as the ag gregation and growth of the calcium oxalate crystals in the system. When the cr ystals are aggregated or grow to a larger size, they will settle down faster due to the heavie r weight. Albumin is also a macromolecule with molecular weight approximately 67 kDa; therefore it is possible to settle down after a period of time. Thus, when albumin binds with calcium oxalate crystals, the settling down effect of albumin will also influence the settling down rate of calcium oxalate crystals as well. Interestingly, the COD crystals structure is also found with the pres ence of albumin [34]. The COD structure is also found in the presence of Khella extract in our expe riments. Therefore, Khella extract might also have an organic macr omolecule that has anionic charge like albumin but has smaller molecular weight so it is not settling down in aqueous solution.

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106 Cell retention studies From the interaction force measurement between a COM crystal and MDCK cells, the experiment in AUIS solution with 5 g/ml of albumin did not show significant changes in maximum adhesion force (Figure 4-37). -0.5 0 0.5 1 1.5 2 050010001500200025003000 Indentation Distance (nm)F (nN) Control Albumin Figure 4-37 The effect of albumin on interact ion force between COM cr ystal and MDCK cells. The combination effect of albumin and oxalate was also measured. Three levels of oxalate and albumin were used to investigate the inte raction effect between species (Table 4-14). Table 4-14 Concentration of albumin and oxala te in AUIS solution used for AFM short range interaction force measurement Solution Albumin (ppm) Oxalate (mM) 1 5 0.1 2 5 10 3 15 0.1 4 15 10 5 10 5

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107 Figure 4-38 Short range inter action force/indention distance curve of MDCK cell and COM crystals. Solution 1-5 albumin and oxalate concentration in AUIS solution shown in Table 4-7. A shorter range of indentation distance was measured in this experiment. It is suspected that the MDCK cells used had lost their epithel ial membrane structure a nd were left with only basement membrane on the substrate. Oxalate is known to be one of the key components that cause the kidney epithelial cell to rupture. When injured or killed, the cells lose their polarity which leads to membrane degradation and expos ition of basement membrane. Therefore, the oxalate content of the solution may have injure d the MDCK cells. If basement membrane was present on the substrate this woul d reduce the indentation distance. Figure 4-38 shows that at highe r concentrations of albumin the short range repulsive forces increased (line 3 and 4). This increase of repulsive for ces might be due to the steric

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108 repulsion from albumin. Albumin pr otein could be considered as a polymer molecule. At higher concentration, albumin might act as a polymeric stabilizer between th e COM crystal and the MDCK cells. The protein might adsorb on the MD CK cell membrane and form a mushroom or packed brush boarder region on the surface, t hus causing steric repulsion. Higher oxalate concentration reduces the repulsive force betw een COM crystal and MDCK cells, due to more injury of the cell from oxalate. When the cell is injured, the cell exposes the anionic-charged phospholipids from the inside layer of the memb rane. The presence of anionic phospholipids at the site of cell injury attracts COM crystals by electrostatic force.

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109 CHAPTER 5 CONCLUSION AND FUTURE WORK Conclusion In this study, the effect of Khella ( Ammi visnaga) on calcium oxalate crystallization was investigated. The light absorbance graph was used to estimate the induction time of nucleated crystals in calcium and oxalate solutions with the addition of extract and ot her species of interest. Addition of Khella extract, sign ificantly prolongs induction time of nucleation which implies that the system requires longer time to form detectab le crystals. Also, the i nhibition of nucleation of the extract was studied using the maximum increasing slope of the light absorbance measurements. It showed that both Khella extr acts studied, Egyptian and Turkish, succeeded in inhibiting nucleation with an average value of 75%. Thus, besides reduc ing the pain of the trapped stones by relaxing muscles, Khella ex tract hinders the nuclea tion of calcium oxalate crystals, the main crystallin e components of the stones. Crystals of the calcium oxalate crystals formed with the presence of extract are mostly in the COD form and some other less sharp-edged crys talline form of calcium oxalate. The crystals formed in the control solution, a mixture of calcium and oxalate solution alone, were purely well-structured COM crystals. This change in crystal hydrate structure from COM to COD from the addition of Khella extract is also beneficial to the kidney stone formers. COD crystals have higher solubility and thermodynamically less st able. The COD structure of calcium oxalate crystal is also believed to dissolve easier than COM crystals. Also, with smoother edge, the COD crystals, cause less physical damage to the kidney epithelial cells. Khella extract also shows that it has a prev ention effect on the COM crystal adhesion to MDCK kidney epithelial cells. Hence, Khella ex tract is effective in kidney stone prevention by

PAGE 110

110 easing down the trapped stone th rough the renal tubule, slows down nucleation of the main crystals of the stone, and re duces the cell adhesion of calci um oxalate monohydrate crystals. Khella extract components were identified us ing several techniques. Our study finds that Khella extract received from Turkey has khellin and visnagin as stat ed in the literature. However, the Egyptian Khella extract does not show appreciable khellin and visnagin in both TLC and HPLC results. This absence of khe llin and visnagin in Egyptian Kh ella extract might be a result from poor storage of the dried seed. Khellin and visnagin might degrade due to humidity, temperature gradient and light exposure. The pure standards of khellin and visnagin were tested on nucleation studies. Using statis tical design of experiments, it was shown that both khellin and visnagin have no statistically significant effect on the induction time a nd percent of nucleation inhibition. The crystals that are formed with the presence of khell in and visnagin are clearly of COM structure and show no sign of other hydrate forms. However, both extracts, Egyptian and Turkish, are found to have an effect, altering morphology and hinde ring nucleation. Interestingly, the HPLC results show a matching unknown peak for both extracts at a retention time of approximately 19 minutes. This organic componen t might be the cause for preventive action on hindering stone formation of Khella extract. This unknown peak should be separated using an HPLC separation column, tested on its nucleation effect, and then identified. Other inorganic components that are suspected to have an influence on calcium oxalate crystallization were investigated. Our results show that Khella extract also contained calcium, magnesium and potassium ions. Calcium ion is one of the main components for nucleating calcium oxalate crystals. Ther efore, addition of calcium i on from extract should promote nucleation of calcium oxalate crystals by incr easing supersaturation. Our groups previous studies, Jeons thesis, sh ow that the high calcium ratio induces nucleation, or has faster induction

PAGE 111

111 time. However, the calcium oxalate crystal mo rphology with high calcium ratio is altered to smoother rounder edges as seen in Figure 4.15. There was, however, no evidence of COD crystals and it was concluded that the high calcium ratio does not contribute to the presence of COD crystals in solutions with extract. The effect of magnesium on the nucleation studies was also investigated. Magnesium has no significan t effect on retardation of calcium oxalate nucleation and crystal morphology. The direct force measurement of COM crys tal and MDCK cell inte raction by AFM shows adhesion force with the presence of both calcium and magnesium in TBS solution. In contrast, the addition of extract sh ows no adhesion force at all. Theref ore, the effect of Khella on cell retention by preventing adhesion between COM and kidney epithelia l cells is not influenced from the content of calcium and magnesium in the extract. The extract organic components, such as khellin and visnagin or the unknown compon ents, might prevent the COM crystals from adhering to the kidne y epithelial cell. Citrate is also a known calcium oxalate nucleation inhibitor. The induction time experiments in our study confirmed that citrate retards induction time at every supersaturation. Citrate is an anionic compound which competitiv ely binds calcium into a complex solution. However, citrate does not nucleate COD crystals. The crystals of calcium oxalate formed with citrate were shown to have th inner and rounder edges of COM. But, they were not COD. The adhesion force measurement in the pres ence of citrate shows a lower adhesion force between COM and MDCK cells. However, the change is small and the adhesion force still exists unlike what we saw with Khella extract. Cellular membrane debris was also studied fo r its effect on nucleation of calcium oxalate crystals. The presence of cellular membrane debris induces nucl eation by changing the

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112 nucleating system from homogeneous to heteroge neous. Using the slope of light absorbance, it was shown that the addition of cell membrane de bris promotes calcium oxalate nucleation. By providing a site for nucleation, th e system requires less energy to precipitate crystals. However, the assumption using classical homog eneous nucleation is not valid in this system since values using homogeneous nucleation theory do not prov ide a good correlation with the data from the experiments. The system with cellular membrane debris should have lower surface energy, less critical energy barriers than th e system without the membrane. However, the results show no change in surface energy and higher critical energy barrier at every supersaturation. Albumin was used to test the effect of urin ary proteins on calcium oxalate nucleation. It was found that albumin inhibits calcium oxalate nucleation by both increasing induction time and percent nucleation inhibition. However, this effect is insignificant when considering the error bar and comparing to the results from Khella extract. Although, albumin is found to have no significant effect on nucleation of calcium oxalate crystals, it is found to promote crystal aggregation. The aggregation of calcium oxalate crystals fr om albumin is due to protein binding to the calcium rich phase of crystals a nd to the settling down e ffect of the protein itself. From the cell retention studies using AFM, it was shown that there is no change in adhesion force with addition of albumin on the long range forces. Howe ver, when measuring the adhesion force with a mixture of albumin and oxalate, a short range force was measured due to MDCK cell losing its epithelial membrane from exposure to oxalate. Th e short range force represented the interaction between COM crystal and the basement membrane of the MDCK cell. It was found that the high albumin level provides higher repulsive fo rce between COM crysta l and the basement membrane. This increasing repulsion c ould be due to steric inhibition.

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113 Future Work Here are some suggestions for future work: Test the effect of pure khellin and vi snagin on the AFM force measurements. Test the effect of extract and other co mponents on crystal-cell adhesion using the 14C COM crystals attachment assay. With AFM, perform the elasticity measurement of the cell with and without extract. Verify crystal structure and do the quantitative analysis of each structure formed using XRD and FTIR. Determine the crystal size distribution of the crystal formed using Coulter particle size analysis. Study the effect of extract on calcium oxalate nucleation in artifi cial and human urine environment. Study the effect of Khella extract on calcium oxalate crystal aggr egation using AFM to measure crystal-crystal interaction force. But, of course, the major objective of future work should be to: Separate the other unknown organic compone nts using HPLC and identify the unknowns. Test the effect of unknown organic com ponents on nucleation and cell retention.

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114 APPENDIX A EFFECT OF KHELLA EXTRACT ON LI GHT ABSORBANCE MEASUREMENT -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 020040060080010001200140016001800Time (s)Absorbance Control 1mg/ml Egyptian extract 1 mg/ml Turkish extract Control induction time with Turkish Khella extract induction time Figure A-1 Light absorbance measurement with the presence of Khella extr act at supersaturation 2.0 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 020040060080010001200140016001800Time (s)Absorbance Control 1mg/ml Egyptian extract 1 mg/ml Turkish extract Control induction time with Turkish Khella extract induction time with EgyptianKhella extract induction time Figure A-2 Light absorbance measurement with the presence of Khella extr act at supersaturation 2.2

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115 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 020040060080010001200140016001800 Time (s)Absorbance Control 1 mg/ml Egyptian extract 1 mg/ml Turkish extract Control induction time with Turkish and Egyptian Khella extract Induction time Figure A-3 Light absorbance measurement with the presence of Khella extr act at supersaturation 2.4 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 020040060080010001200140016001800Time (s)Absorbance Control 1mg/ml Egyptian extract 1 mg/ml Turkish extract withTurkish Khella extract induction time with Egyptian Khella extract induction time Control induction time Figure A-4 Light absorbance measurement with the presence of Khella extr act at supersaturation 2.6

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116 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 020040060080010001200140016001800 Time (s)Absorbance Control 1mg/ml Egyptian extract 1 mg/ml Turkish extract with Turkish Khella extract induction time with Egyptian Khella extract induction time Control induction time Figure A-5 Light absorbance measurement with the presence of Khella extr act at supersaturation 2.8

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117 APPENDIX B EFFECT OF KHELLA ON THE CHANGE IN GIBBS FREE ENERGY OF FORMATION -1E-20 -5E-21 0 5E-21 1E-20 1.5E-20 2E-20 2.5E-20 0.00.10.20.30.40.50.60.70.80.91.0Radius (nm)Change in Gibbs free energy of formation (J) Control with Egyptian Khella extract with Turkish Khella extract Figure B-1 Effect of Khella extract on GN at initial supersat uration of SS 2.0 -1E-20 -5E-21 0 5E-21 1E-20 1.5E-20 0.000.100.200.300.400.500.600.700.80Radius (nm)Change in Gibbs free energy of formation (J) Control with Egyptian Khella extract with Turkish Khella extract Figure B-2 Effect of Khella extract on GN at initial supersat uration of SS 2.2

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118 -2E-20 -1.5E-20 -1E-20 -5E-21 0 5E-21 1E-20 1.5E-20 00.10.20.30.40.50.60.70.8Radius (nm)Change in Gibbs free energy of formation (J) Control w/Egyptian Khella extract w/ Turkish Khella extract Figure B-3 Effect of Khella extract on GN at initial supersat uration of SS 2.4 -6E-21 -4E-21 -2E-21 0 2E-21 4E-21 6E-21 8E-21 1E-20 00.10.20.30.40.50.6Radius (nm)Change in Gibbs free energy of formation (J) Control with Egyptian Khella extract with Turkish Khella extract Figure B-4 Effect of Khella extract on GN at initial supersat uration of SS 2.6

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119 -6E-21 -4E-21 -2E-21 0 2E-21 4E-21 6E-21 8E-21 1E-20 00.050.10.150.20.250.30.350.40.450.5Radius (nm)Change in Gibbs free energy of nucleation (J) Control with Egyptian Khella extract with Turkish Khella extract Figure B-5 Effect of Khella extract on GN at initial supersat uration of SS 2.8

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120 APPENDIX C AFM MEASUREMENTS OF COM CRYSTAL AND MDCK CELLS INTERACTION FORCE -1 0 1 2 010002000 Distance ( nm ) Force (nN) Figure C-1 Example of AFM measur ements of COM-MDCK cells interaction in artificial urine solution with 1 mg/ml of Khella extract. -1 0 1 2 0100020003000 Distance (nm)Force (nN) Figure C-2 Example of AFM measur ements of COM-MDCK cells interaction in artificial urine solution.

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121 APPENDIX D EFFECT OF KHELLIN AND VISNAGIN PURE COMPOUNDS ON LIGHT ABSORBANCE MEASUREMENT -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 020040060080010001200140016001800Time (s)AbsorbanceStandard # 5, 10, 12, 13 Standard # 11 Figure D-1 Light absorbance measurement of standard # 5, 10, 11, 12, 13. (0.015 mg/ml Khellin and 0.0025 mg/ml visnagin) -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 020040060080010001200140016001800Time (s)Absorbance Control with 0.015 mg/ml Khellin with 0.03 mg/ml Khellin Induction time with Khellin Control induction timeStandard # 1 Standard # 2 Standard # 3 Figure D-2 Effect of khellin on light absorban ce measurement (standard # 1, 2, and 3).

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122 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 020040060080010001200140016001800Time (s)Absorbance Control with 0.0025 mg/ml visnagin with 0.005 mg/ml visnagin with 0.0025 mg/ml visnagin induction time Control induction time with 0.005 mg/ml visna g in induction time Figure D-3 Effect of visnagin on light absorb ance measurement (standard # 5, 1, and 4). -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 020040060080010001200140016001800Time (s)AbsorbanceStd #9Std #10-13 Std #7 Std #6 Std #2,8 Std #3 Std #4 Std #5 Std #1 Figure D-4 Comparison of light absorbance m easurement of all standard run number.

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123 APPENDIX E ANOVA FOR % NUCLEATION INHIBITION RESPONSE SURFACE OF KHELLIN AND VISNAGIN PURE COMPOUND A = Khellin B = Visnagin Response:% Nucleation Inhibition ANOVA for Response Surface 2FI Model Table E-1 Analysis of variance table [Partial sum of squares] Source Sum of Squares DF Mean Square F Value Prob > F Model 2151.29 3 717.1 5.39 0.0253 significant A 62.29 1 62.29 0.47 0.513 B 864 1 864 6.5 0.0342 AB 1225 1 1225 9.21 0.0162 Residual 1063.71 8 132.96 Lack of Fit 780.51 4 195.13 2.76 0.1749 not significant Pure Error 283.2 4 70.8 Cor Total 3215 11 The Model F-value of 5.39 implies the model is signi ficant. There is only a 2.53% chance that a "Model F-Value" this large could occur due to no ise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case B, AB are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve your model. The "Lack of Fit F-value" of 2.76 implies the La ck of Fit is not significant relative to the pure error. There is a 17.49% chance that a "Lack of F it F-value" this large could occur due to noise. Non-significant lack of fit is good -we want the model to fit. Table E-2 Statistic results Std. Dev. 11.53 R-Squared 0.6691 Mean 7.50 Adj R-Squared 0.5451 C.V. 153.75 Pred R-Squared 0.1747 PRESS 2653.28 Adeq Precision 8.862 The "Pred R-Squared" of 0.1747 is not as close to the "Adj R-Squared" of 0.5451 as one might normally expect. This may indicate a large bloc k effect or a possible problem with your model and/or data. Things to consid er are model reduction, response tr anformation, outliers, etc. "Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 8.862 indicates an adequate signal. This model can be us ed to navigate the design space.

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124 Final Equation in Terms of Coded Factors: Nucleation Inhibition = +7.20 +3.56 A -12.00 B -17.50 A B Final Equation in Terms of Actual Factors: Nucleation Inhibition = -1.85593 +1403.95480 Khellin +2200.00000 Visnagin -4.66667E+005 Khellin Visnagin Table E-3 Diagnostics case statistics Standard Order Actual Value Predicted Value ResidualLeverage Student Residual Cook's Distance Outlier t Run Order 1 0 -1.86 1.86 0.739 # 0.315 0.07 0.296 5 2 27 19.2 7.8 0.251 0.781 0.051 0.761 13 3 37 40.26 -3.26 0.671 # -0.493 0.124 -0.469 9 5 -1 7.2 -8.2 0.085 -0.744 0.013 -0.721 12 6 21 10.76 10.24 0.254 1.028 0.09 1.032 7 7 5 9.14 -4.14 0.739 # -0.703 0.349 -0.679 4 8 15 -4.8 19.8 0.251 1.984 0.331 2.605 6 9 -28 -18.74 -9.26 0.671 # -1.4 0.999 -1.508 3 10 3 7.2 -4.2 0.085 -0.381 0.003 -0.36 10 11 17 7.2 9.8 0.085 0.888 0.018 0.875 8 12 -3 7.2 -10.2 0.085 -0.925 0.02 -0.916 11 13 -3 7.2 -10.2 0.085 -0.925 0.02 -0.916 2 # Obs with Leverage > 2.00 *(average leverage) Proceed to Diagnostic Plots (the next icon in progression). Be sure to look at the: 1) Normal probability plot of the studentiz ed residuals to check for normality of residuals. 2) Studentized residuals versus predic ted values to check for constant error. 3) Outlier t versus run order to lo ok for outliers, i.e., influential values. 4) Box-Cox plot for power transformations. If all the model statistics a nd diagnostic plots are OK, finish up with the Model Graphs icon.

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125 APPENDIX F EFFECT OF MAGNESIUM ON LIGHT ABSORBANCE MEASUREMENT -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 020040060080010001200140016001800Time (s)Absorbance Control with 0.1mM Magnesium with 0.1mM Magnesium induction time Control induction time Figure F-1 Effect of magnesium on light absorbance measurement at supersaturation 2.0 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 020040060080010001200140016001800Time (s)Absorbance Control with 0.1mM Magnesium Control induction time with 0.1mM Magnesium induction time Figure F-2 Effect of magnesium on light absorbance measurement at supersaturation 2.2

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126 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05 020040060080010001200140016001800Time (s)Absorbance Control with 0.1mM Magnesium Control & with 0.1 mM Magnesium induction time Figure F-3 Effect of magnesium on light absorbance measurement at supersaturation 2.6 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 020040060080010001200140016001800Time (s)Absorbance Control with 0.1mM Magnesium Control induction time with 0.1mM Magnesium induction time Figure F-4 Effect of magnesium on light absorbance measurement at supersaturation 2.8

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127 APPENDIX G EFFFECT OF CITRATE ON LIGH T ABSORBANCE MEASUREMENT -0.005 0 0.005 0.01 0.015 0.02 0.025 020040060080010001200140016001800Time (s)Absorbance Control with 0.1mM Citrate Control induction time with 0.1mM Citrate induction time Figure G-1 Effect of citrate on light absorb ance measurement at supersaturation 2.0 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 020040060080010001200140016001800Time (s)Absorbance Control with 0.1mM Citrate Control induction time with 0.1mM Citrate induction time Figure G-2 Effect of citrate on light absorb ance measurement at supersaturation 2.2

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128 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05 020040060080010001200140016001800Time (s)Absorbance Control with 0.1mM Citrate Control induction time with 0.1mM Citrate induction time Figure G-3 Effect of citrate on light absorb ance measurement at supersaturation 2.6 -0.005 0.005 0.015 0.025 0.035 0.045 0.055 0.065 020040060080010001200140016001800Time (s)Absorbance Control with 0.01mM Citrate Control induction time with 0.1mM Citrate induction time Figure G-4 Effect of citrate on light absorb ance measurement at supersaturation 2.8

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129 APPENDIX H EFFECT OF CELLULAR MEMBRANE DEBRIS ON LIGHT ABSORBANCE MEASUREMENT -0.002 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0.02 05010015020025030035040 Time (s)Absorbance Control with MDCK cell debris with LLC-PK1 cell debris with MDCK debris induction time with LLC-PK1 debris induction time Control induction time Figure H-1 Effect of cellular membrane de bris on light absorbance measurement at supersaturation 2.0 -0.005 0 0.005 0.01 0.015 0.02 0.025 050100150200250300350400 Time (s)Absorbance Control with MDCK cell debris with LLC-PK1 cell debris Control induction time with MDCK debris induction time with LLC-PK1 induction time Figure H-2 Effect of cellular membrane de bris on light absorbance measurement at supersaturation 2.2

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130 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 050100150200250300350400 Time (s)Absorbance Control with MDCK cell debris with LLC-PK1 cell debris Control induction time with MDCK induction time with LLC-PK1 induction time Figure H-3 Effect of cellular membrane de bris on light absorbance measurement at supersaturation 2.4 -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 050100150200250300350400Time (s)Absorbance Control with MDCK cell debris with LLC-PK1 cell debris with LLC-PK1 and MDCK debris induction tiem Control induction time Figure H-4 Effect of cellular membrane de bris on light absorbance measurement at supersaturation 2.8

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131 -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 050100150200250300350400Time (s)Absorbance Control with MDCK cell debris with LLC-PK1 cell debris induction time are relatively the same. Figure H-5 Effect of cellular membrane de bris on light absorbance measurement at supersaturation 3.0 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 050100150200250300350400Time (s)Absorbance Control with MDCK cell debris with LLC-PK1 cell debris induction time is relatively the same Figure H-6 Effect of cellular membrane de bris on light absorbance measurement at supersaturation 3.2

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132 APPENDIX I EFFECT OF CELLULAR MEMBRANE DEBRIS ON CHANGE IN GIBBS FREE ENERGY OF FORMATION -1E-21 1E-21 3E-21 5E-21 7E-21 9E-21 1.1E-20 1.3E-20 1.5E-20 00.10.20.30.40.50.60.7Radius (nm)Change in Gibbs free energy of formation (J) Control with LLC-PK1 cell debris with MDCKcell debris Figure I-1 Effect of cellular membrane on the change in Gibbs free energy of formation at supersaturation 2.0 -1E-21 1E-21 3E-21 5E-21 7E-21 9E-21 1.1E-20 1.3E-20 1.5E-20 00.10.20.30.40.50.60.7Time (s)Change in Gibbs free energy of formation (J) Control with LLC-PK1 cell debris with MDCK cell debris Figure I-2 Effect of cellular membrane debris on change in Gibbs free energy of formation at supersaturation 2.2

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133 -1E-21 1E-21 3E-21 5E-21 7E-21 9E-21 00.10.20.30.40.50.6Radius (nm)Change in Gibbs Free Energy (J) Control with LLC-PK1 cell debris with MDCK cell debris Figure I-3 Effect of cellular membrane debris on change in Gibbs free energy of formation at supersaturation 2.4 -1E-21 1E-21 3E-21 5E-21 7E-21 9E-21 00.10.20.30.40.50.6Time (s)Change in Gibbs free energy of formation (J) Control with LLC-PK1 cell debris with MDCK cell debris Figure I-4 Effect of cellular membrane debris on change in Gibbs free energy of formation at supersaturation 2.6

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134 -1E-21 1E-21 3E-21 5E-21 7E-21 9E-21 00.050.10.150.20.250.30.350.40.450.5Time (s)Change in Gibbs free energy of formation (J) Control with LLC-PK1 cell debris with MDCK cell debris Figure I-5 Effect of cellular membrane debris on change in Gibbs free energy of formation at supersaturation 2.8

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135 APPENDIX J EFFECT OF ALBUMIN PROTEINS ON LIGHT ABSORBANCE MEASUREMENT -0.004 -0.002 0 0.002 0.004 0.006 0.008 0.01 0.012 020040060080010001200140016001800Time (s)Absorbance Control with Albumin Control induction time with Albumin induction time Figure J-1 Light absorbance measurement with the effect of albumin prot ein at supersaturation 2.0. -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 020040060080010001200140016001800Time (s)Absorbance Control with Albumin Control induction time with Albumin induction time Figure J-2 Light absorbance measurement with the effect of albumin prot ein at supersaturation 2.2.

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136 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 020040060080010001200140016001800Time (s)Absorbance Control with Albumin Control induction time with Albumin induction time Figure J-3 Light absorbance measurement with the effect of albumin prot ein at supersaturation 2.4. -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 020040060080010001200140016001800Time (s)Absorbance Control with Albumin Control induction time with Albumin induction time Figure J-4 Light absorbance measurement with the effect of albumin prot ein at supersaturation 2.8.

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141 BIOGRAPHICAL SKETCH Saijit Daosukho was born in Bangkok, Thailand. She attended Kasetsart University Laboratory School from grades 1 to 12. Saijit received a scholars hip from the Thai Government after graduating from high school to study in part icle engineering. She spent one year as a post graduate student at Wyoming Seminary College Preparation School in Kingston, Pennsylvania. After which, she attended Carnegie Mellon Univer sity in Pittsburgh, Pennsylvania and obtained her B.S. in materials science and engineering wi th minor in electronic materials in August 2001. She began her graduate studies in material s science and engineer ing department at University of Florida and received her Master of Science in December, 2003. There, she started researching particle interaction and became involve d in a kidney stone project under supervisory of Dr. Hassan El-Shall. After co mpleting her Masters degree, Sa ijit transferred to chemical engineering department to pur sue her Doctor of Philosophy degree in 2004. However, she continued working on her kidney stone project u nder co-supervision of Dr. Spyros Svoronos and Dr. Hassan El-Shall. Once she graduates from the University of Florida in December 2007, she will begin work as a research scie ntist for the Scien ce, Technology, and Environmenta l Ministry of Thailand.